Characterization of Klebseilla pneumoniae T6SS nanoweapon and its role in the dissemination of antimicrobial genes and virulence factors.

Our struggle against infectious diseases is far from over. Globalisation has increased the risk of pandemics, and the rise of antibiotic-resistant microbes threatens to render existing drugs useless. Of particular concern is the health burden of respiratory infections being the UK in the top 25 countries for deaths from acute respiratory infections, above most other European countries. Of great concern is the mounting prevalence of respiratory infections caused by Gram-negative bacteria, in particular Klebsiella pneumoniae (the focus of this project). Worryingly, there are reports showing a 15% increased in incidence of Klebsiella infections in the last five years only in the UK. This is particularly alarming given the high rates of resistance to empirical antibiotics commonly recommended for Klebsiella treatment. More than a third of the K. pneumoniae isolates reported to the European Centre for Disease Prevention and Control were resistant to at least one antimicrobial group, being the most common resistance phenotype the combined resistance to fluoroquinolones, third-generation cephalosporins and aminoglycosides. Of note, in Klebsiella, like in other microbes, the transfer of antimicrobial resistance genes occurs via sharing plasmids between different microbes. Epidemiological studies have demonstrated that Klebsiella strains have access to a mobile pool of antimicrobial genes and virulence factors, enabling the emergence of a multidrug, hypervirulent K. pneumoniae clone capable of causing untreatable infections in healthy individuals. Not surprisingly, Klebsiella has been singled out as an "urgent threat to human health" by the UK Government, the U.S. Centers for Disease Control and Prevention, and the World Health Organization due to extremely drug resistant strains.
Recently, the Bengoechea laboratory has demonstrated that K. pneumoniae employs a nanowaepon, the type VI secretion system (T6SS), to antagonize other microbes. Therefore, the antimicrobial action of the T6SS is at odds with the dissemination of antimicrobial genes and virulence factors. In this project, our research will uncover how Klebsiella resolves the conflict between the transfer of genetic material and the activity of the T6SS. We will also provide new insights into the T6SS assemblies that Klebsiella produces to deliver the antimicrobial toxins to other bacteria, and identify the portfolio of toxins that Klebsiella deploys. We will also highlight a hitherto unknown connection between the T6SS and the lipopolysaccharide, a glycolipid located in the surface of all Gram-negative bacteria.
A better understanding of the barriers and limitations of the transfer of material between microbes is invaluable to predict outbreaks of resistant microbes in the health care setting, and may result in developing new therapeutics to limit the process. In addition, our research will provide a global vision of the antimicrobial strategies deployed by Klebsiella. Finding new toxins and determining their mechanisms of action shall be a gold mine of usable antibacterial targets that pharmaceutical companies could consider to develop much needed new drugs against multidrug resistant infections.

Klebsiella pneumoniae has been singled out as a threat to human health due to the increasing number of multidrug resistant strains. Alarmingly, recent studies have recognized that K. pneumoniae strains have access to a mobile pool of virulence genes; enabling the emergence of a multidrug, hypervirulent species. Unfortunately, there are already reports describing the isolation of such strains. We have recently uncovered that Klebsiella exploits a type VI secretion system (T6SS) for contact-dependent killing of neighboring, non-sister bacterial competitors. T6SS activity possess a unique challenge to the dissemination of mobile genetic elements encoding virulence genes and antibiotic resistance via conjugation. Based on solid preliminary data, we will provide mechanistic insights into how Klebsiella resolves the conflict between transfer and acquisition of genetic material, and the function of the T6SS. This proposal will establish that bacteria switch the T6SS behavior from offensive to defensive to facilitate conjugation and the resulting dissemination of antimicrobial genes and virulence factors. We will also highlight previous unreported existence of distinct versions of the T6SS machinery, characterized by a specific set of Hvp-VgrGs pairs, to deploy effectors. By a quantitative proteomic approach, we will characterize the repertoire of T6SS effectors secreted by each assembly, and provide mechanistic insights into the antimicrobial function of T6SS effectors. In this project we will also establish a hitherto unknown link between the T6SS and the lipopolysaccharide (LPS). Our work will uncover the regulatory network connecting these two major structures of the envelope of Gram-negative bacteria, shedding new light into the inputs and outputs governing the bacterial cell wall. The findings of this project will establish basic principles of the T6SS while providing a global vision of Klebsiella's T6SS-governed antimicrobial strategy.