A Genomic Approach To Identify Klebsiella pneumoniae and Acinetobacter baumannii Strains with Enhanced Competitive Fitness in the Lungs during Multistrain Pneumonia.

Microbial competition is most often studied at the genus or species level, but interstrain competition has been less thoroughly examined. Klebsiella pneumoniae is an important pathogen in the context of hospital-acquired pneumonia, and a better understanding of strain competition in the lungs could explain why some strains of this bacterium are more frequently isolated from pneumonia patients than others. We developed a barcode-free method called "StrainSeq" to simultaneously track the abundances of 10 K. pneumoniae strains in a murine pneumonia model. We demonstrate that one strain (KPPR1) repeatedly achieved a marked numerical dominance at 20 h postinoculation during pneumonia but did not exhibit a similar level of dominance in in vitro mixed-growth experiments. The emergence of a single dominant strain was also observed with a second respiratory pathogen, Acinetobacter baumannii, indicating that the phenomenon was not unique to K. pneumoniae When KPPR1 was removed from the inoculum, a second strain emerged to achieve high numbers in the lungs, and when KPPR1 was introduced into the lungs 1 h after the other nine strains, it no longer exhibited a dominant phenotype. Our findings indicate that certain strains of K. pneumoniae have the ability to outcompete others in the pulmonary environment and cause severe pneumonia and that a similar phenomenon occurs with A. baumannii In the context of the pulmonary microbiome, interstrain competitive fitness may be another factor that influences the success and spread of certain lineages of these hospital-acquired respiratory pathogens.

A novel phosphatidylinositol 4,5-bisphosphate binding domain mediates plasma membrane localization of ExoU and other patatin-like phospholipases.

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains.

PGE(2) suppression of innate immunity during mucosal bacterial infection.

Prostaglandin E2 (PGE2) is an important lipid mediator in inflammatory and immune responses during acute and chronic infections. Upon stimulation by various proinflammatory stimuli such as lipopolysaccharide (LPS), interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α, PGE2 synthesis is upregulated by the expression of cyclooxygenases. Biologically active PGE2 is then able to signal through four primary receptors to elicit a response. PGE2 is a critical molecule that regulates the activation, maturation, migration, and cytokine secretion of several immune cells, particularly those involved in innate immunity such as macrophages, neutrophils, natural killer cells, and dendritic cells. Both Gram-negative and Gram-positive bacteria can induce PGE2 synthesis to regulate immune responses during bacterial pathogenesis. This review will focus on PGE2 in innate immunity and how bacterial pathogens influence PGE2 production during enteric and pulmonary infections. The conserved ability of many bacterial pathogens to promote PGE2 responses during infection suggests a common signaling mechanism to deter protective pro-inflammatory immune responses. Inhibition of PGE2 production and signaling during infection may represent a therapeutic alternative to treat bacterial infections. Further study of the immunosuppressive effects of PGE2 on innate immunity will lead to a better understanding of potential therapeutic targets within the PGE2 pathway.

Post-exposure therapeutic efficacy of COX-2 inhibition against Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative, facultative intracellular bacillus and the etiologic agent of melioidosis, a severe disease in Southeast Asia and Northern Australia. Like other multidrug-resistant pathogens, the inherent antibiotic resistance of B. pseudomallei impedes treatment and highlights the need for alternative therapeutic strategies that can circumvent antimicrobial resistance mechanisms. In this work, we demonstrate that host prostaglandin E2 (PGE2) production plays a regulatory role in the pathogenesis of B. pseudomallei. PGE2 promotes B. pseudomallei intracellular survival within macrophages and bacterial virulence in a mouse model of pneumonic melioidosis. PGE2-mediated immunosuppression of macrophage bactericidal effector functions is associated with increased arginase 2 (Arg2) expression and decreased nitric oxide (NO) production. Treatment with a commercially-available COX-2 inhibitor suppresses the growth of B. pseudomallei in macrophages and affords significant protection against rapidly lethal pneumonic melioidosis when administered post-exposure to B. pseudomallei-infected mice. COX-2 inhibition may represent a novel immunotherapeutic strategy to control infection with B. pseudomallei and other intracellular pathogens.