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. In 1890, Robert Koch formulated Koch's postulates as general guidelines for identifying disease-causing organisms. One century later, Stanley Falkow established the molecular version of Koch's postulates, this time to guide the identification of microbial genes encoding virulence factors. One of the key points of the molecular postulates is to test the virulence of a microorganism with an inactivated candidate virulence gene in an appropriate animal model. Therefore, the use of animal models to identify the virulence factors of human pathogens is indispensable. Currently, identification and characterization of novel virulence factors rely largely on assessing mutant bacteria for growth in the organs of infected mice. The dependence on mouse infection models limits large-scale analysis of virulence due to the large number of animals needed to obtain statistically significant results.To circumvent these issues, the search for alternative host models is ongoing. Ideally, these alternative models should be easy to maintain and infect, should be amenable to genetic manipulation, and should model aspects of vertebrate defenses upon infection, chiefly the immune response. The immune defense consists of two main parts, an innate and an adaptive response, wit...

Klebsiella pneumoniae is an important cause of multidrug‐resistant infections worldwide. Recent studies highlight the emergence of multidrug‐resistant K. pneumoniae strains which show resistance to colistin, a last‐line antibiotic, arising from mutational inactivation of the mgrB regulatory gene. However, the precise molecular resistance mechanisms of mgrB‐associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an mgrB gene K. pneumoniae mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mgrB mutation induces PhoPQ‐governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances K. pneumoniae virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence.

This is the peer reviewed version of the following article: Cano, V., March, C., Insua, J. L., Aguilo, N., Llobot, E., MorataD., Regueiro, V., Brenan, G. P., Millan-Lou, M. I., Martin, C., Garmendia, J., Bengoechea, J. A. (2015),Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes. Cellular Microbiology, VOL: PAGE NO's. which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/cmi.12466/abstract. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. General rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights.Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk.

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Background: There is limited knowledge of Klebsiella pneumoniae determinants implicated in the blocking of the NF-κB signaling pathway.Results: A high-throughput genetic screen led to the identification of 114 putative K. pneumoniae genes that are associated with suppression of NF-κB activation.Conclusion: CPS, LPS, and the T2SS-secreted PulA are needed for immune evasion.Significance: A new therapeutic approach to treat Klebsiella infections will be the prevention of immune evasion.