No abstract

Worldwide, Klebsiella pneumoniae is an increasingly problematic opportunistic pathogen, with the emergence of carbapenem-resistant isolates of special importance. The mechanisms of virulence are poorly understood, and the current study utilized the invertebrate model Galleria mellonella to investigate facets of the virulence process. A range of UK clinical isolates and reference strains was assessed in Galleria by measuring survival as an end point. The clinical strains showed a range of virulence, with the majority of strains (68 %) causing greater than 50 % mortality at a challenge dose of 1¾10 5 c.f.u. Three additional intermediate read-outs were developed to allow the mechanisms of virulence of Klebsiella to be dissected further. The release of lactate dehydrogenase as a marker of cell damage was the best predictor of virulence. Melanization as a marker of the insect innate immune system and ability to proliferate within Galleria as a marker of immune evasion also broadly correlated with survival but with some notable exceptions. No direct correlation was observed between virulence and either K1 or other defined capsular types, the carriage of defined virulence factors or particular functional phenotypes. Overall, the study showed that Galleria can provide significant insights into the mechanisms of virulence, and that this can be applied to the study of opportunistic human pathogens.

The opportunistic nosocomial pathogen Acinetobacter baumannii is responsible for a growing number of infections; however, few of its potential virulence factors have been identified, and how this organism causes infection remains largely unknown. Bacterial biofilms are often an important component in infection and persistence but there is no conclusive evidence to link biofilm formation with virulence and severity of infection in Acinetobacter. To investigate this link, several clinical isolates were assessed in biofilm culture models and were tested for virulence in the insect model Galleria mellonella. In both systems, the profiles showed significant differences between strains, but no correlation was observed between virulence and the ability to form biofilms. In contrast, A. baumannii cells from a biofilm produced higher mortality rates than an equivalent number of planktonic cells. Relative to planktonic cells, A. baumannii biofilm cultures also showed reduced sensitivity to antibiotics normally used in the treatment of A. baumannii, especially colistin. This model, therefore, provides a suitable system to investigate the link between biofilm growth and various factors influencing virulence during A. baumannii infection.

In the crystal structure of uncomplexed native chymosin, the beta-hairpin at the active site, known as 'the flap', adopts a different conformation from that of other aspartic proteinases. This conformation would prevent the mode of binding of substrates/inhibitors generally found in other aspartic proteinase complexes. We now report the X-ray analysis of chymosin complexed with a reduced bond inhibitor CP-113972 ¿(2R,3S)-isopropyl 3-[(L-prolyl-p-iodo-L-phenylalanyl-S-methyl-cysteinyl)amino-4]-cyclohexy l-2-hydroxybutanoate¿ at 2.3 A resolution in a novel crystal form of spacegroup R32. The structure has been refined by restrained least-squares methods to a final R-factor of 0.19 for a total of 11 988 independent reflections in the resolution range 10 to 2.3 A. The extended beta-strand conformation of the inhibitor allows hydrogen bonds within the active site, while its sidechains make both electrostatic and hydrophobic interactions with residues lining the specificity pockets S4-->S1. The flap closes over the active site cleft in a way that closely resembles that of other previously determined aspartic proteinase inhibitor complexes. We conclude that the usual position and conformation of the flap found in other aspartic proteinases is available to native chymosin. The conformation observed in the native crystal form may result from intermolecular interactions between symmetry-related molecules in the crystal lattice.