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Bengoechea, J. A.

Publications and source records attributed to Bengoechea, J. A..

2 recordsLinked to original sources
bioRxiv2019

A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

The use of animal infection models is essential to understand microbial pathogenesis and to develop and test treatments. Insects, and 2D and 3D tissue models are increasingly being used as surrogate for mammalian models. However, there are concerns whether these models recapitulate the complexity of host-pathogen interactions. Here, we developed the ex vivo lung perfusion (EVLP) model of infection using porcine lungs to investigate Klebsiella pneumoniae-triggered pneumonia as model of respiratory infections. The porcine EVLP model recapitulates features of K. pneumoniae-induced pneumonia lung injury. This model is also useful to assess the pathogenic potential of K. pneumoniae as we observed that the attenuated Klebsiella capsule mutant strain caused less pathological tissue damage with a concomitant decrease in the bacterial burden compare to lungs infected with the wild type. The porcine EVLP model allows assessment of inflammatory responses following infection; similar to the mouse pneumonia model, we observed an increase of il-10 in the lungs infected with the wild type and an increase of ifn-{gamma} in lungs infected with the capsule mutant. This model also allows monitoring phenotypes at the single-cell level. Wild-type K. pneumoniae skews macrophages towards an M2-like state. In vitro experiments probing pig bone marrow-derived macrophages uncovered the role of the M2 transcriptional factor STAT6, and that Klebsiella-induced il10 expression is controlled by p38 and ERK. Klebsiella-induced macrophage polarization is dependent on the capsule. Altogether, this study support the utility of the EVLP model using pig lungs as platform to investigate the infection biology of respiratory pathogens.\n\nIMPORTANCEThe implementation of infection models that approximate human disease is essential to understand infections and for testing new therapies before they enter into clinical stages. Rodents are used in most of pre-clinical studies, although the differences between mouse and man have fuelled the conclusion that murine studies are unreliable predictors of human outcomes. Here, we have developed a whole lung porcine model of infection using the established ex vivo lung perfusion (EVLP) system established to re-condition human lungs for transplant. As a proof-of-principle, we provide evidence demonstrating that infection of the porcine EVLP with the human pathogen K. pneumoniae recapitulates the known features of Klebsiella-triggered pneumonia. Moreover, our data revealed the porcine EVLP model is useful to reveal features of the virulence of K. pneumoniae including the manipulation of immune cells. Altogether, this study supports the utility of the EVLP model using pig lungs as surrogate host for assessing respiratory infections.

microbiology↗
bioRxiv2019

Klebsiella pneumoniae type VI secretion system-mediated microbial competition is PhoPQ controlled and reactive oxygen species dependent

Klebsiella pneumoniae is recognized as an urgent threat to human health due to the increasing isolation of multidrug resistant strains. Hypervirulent strains are a major concern due to their ability to cause life-threating infections in healthy hosts. The type VI secretion system (T6SS) is widely implicated in microbial antagonism, and it mediates interactions with host eukaryotic cells in some cases. In silico search for genes orthologous to T6SS component genes and T6SS effector genes across 700 K. pneumoniae genomes shows extensive diversity in T6SS genes across the K. pneumoniae species. Temperature, oxygen tension, pH, osmolarity, iron levels, and NaCl regulate the expression of the T6SS encoded by a hypervirulent K. pneumoniae strain. Polymyxins and human defensin 3 also increase the activity of the T6SS. A screen for regulators governing T6SS uncover the correlation between the transcription of the T6SS and the ability to kill E. coli prey. Whereas H-NS represses the T6SS, PhoPQ, PmrAB, Hfq, Fur, RpoS and RpoN positively regulate the T6SS. K. pneumoniae T6SS mediates intra and inter species bacterial competition. This antagonism is only evident when the prey possess an active T6SS. The PhoPQ two component system governs the activation of K. pneumoniae T6SS in bacterial competitions. Mechanistically, PhoQ periplasmic domain, and the acid patch within, is essential to activate K. pneumoniae T6SS. Klebsiella T6SS also mediates anti-fungal competition. We have delineated the contribution of each of the individual VgrGs in microbial competition, and identified VgrG4 as a T6SS effector. Structurally, domain DUF2345 of VgrG4 is sufficient to intoxicate bacteria and yeast. ROS generation mediates the antibacterial effects of VgrG4, and the antitoxin Sel1E protects against the toxic activity of VgrG4. Our findings provide a better understanding of the regulation of the T6SS in bacterial competitions, and place ROS as an early event in microbial competition.\n\nAUTHOR SUMMARYKlebsiella pneumoniae has been singled out as an \"urgent threat to human health\" due to extremely drug resistant strains. Numerous studies investigate the molecular mechanisms underlying antibiotic resistance in K. pneumoniae, while others dissect the virulence strategies of this pathogen. However, there is still limited knowledge on the fitness of Klebsiella in the environment, and particularly the competition of Klebsiella with other species. Here, we demonstrated that Klebsiella exploits the type VI secretion system (T6SS) nanoweapon to kill bacterial competitors and fungi. K. pneumoniae perceives T6SS attacks from bacterial competitors, resulting in retaliation against the aggressive cell. The perception of the attack involved the sensor PhoPQ and led to the up-regulation of the T6SS. We identified one of the toxins deployed by the T6SS to antagonize other microbes, and revealed how Klebsiella protects itself from this toxin. Our findings provide a better understanding of the T6SS role in microbial competition and uncover new aspects on how bacteria regulate T6SS-mediated microbial antagonism.

microbiology↗