Limiting infection: Identifying factors for the in vivo dissemination of the major nosocomial pathogen Klebsiella pneumoniae

The zebrafish infection model (Danio rerio) is a reliable vertebrate model which harbors a mammalian-like innate immune system. D. rerio has successfully been used to elucidate factors necessary for host-pathogen interactions1. The success of this model rests on the non-invasive live-cell imaging techniques allowing mechanistic details for tissue-tropism and pathogen-specific survival mechanisms to be established.
In this project, we seek to establish and implement a zebrafish model to study the infective process and the subsequent immune response of the major human pathogen Klebsiella pneumoniae (KPN). KPN is a major nosocomial pathogen, driven by its ability to acquire and transmit antimicrobial resistance (AMR), that according to the CDC and WHO poses a critical threat to human health. As such, there are two main underdeveloped research strategies which require biological and mechanistic validation to limit KPN infection; the first, is detailed systematic high-throughput genome-based analyses of KPN infection and the second, is the exploitation of host-driven diagnostics for rationalisation of antibiotic use. Given that antimicrobial resistance is a critical healthcare issue, the understanding of in-vivo transmission and survival is necessary in the identification, stratification and use of antimicrobial therapy against Klebsiella.
Whilst, studies that describe KPN-D. rerio infection models exist, these do not provide a systematic assessment of both bacterial and host factors necessary for infection. Therefore, the overriding aim of this proposal is to establish and implement an in-vivo infection model to identify the specific roles of key innate cells such as macrophages which can be mapped to specific Klebsiella characteristics. As such, the key aims of this proposal are to firstly, establish the molecular parameters of a Klebsiella-D. rerio in vivo infection model, and secondly, identify specific-innate immune signatures for diagnostic or therapeutic exploitation.
Aim 1: Establish and Implement a Klebsiella-D. rerio infection model system. To implement further studies into the pathogen-host response we will first parameterise with factors such as types of KPN infection routes2 e.g. yolk sac vs otic vesicle, bacterial inoculum, time of colonisation to infection, rates of bacterial transmission with tagged and untagged strains of Klebsiella pneumoniae.
Aim 2: Establish the pathogen factors necessary for KPN infection in D.rerio. With the model developed in Aim 1, where using a Tn-seq approach, transposon mutant pools of candidate KPN strain(s), will be used to establish the genetic factors necessary for in vivo infection. This data is critical in identifying novel genes relevant to infection but also in validating the model against previously identified bacterial factors in murine and human infection models.
Aim 3: Establish the infection dynamics of WT KPN in a macrophage depleted model of D. rerio. The current MRC AMR strategy underscores the exploitation of the innate immune response as a treatment alternative against antibiotic-resistant bacteria. In collaboration with Prof Dockrell and the MRC-funded SHIELD consortium (https://shieldamrresearch.org/about/), we will use the macrophage depleted transgenic D.rerio model3 to characterise the effects of KPN infection. Comparative analyses of data derived from Aim 2and 3 will allow the mapping of specific KPN-macrophage responses and validate pathogen and host factors for systemic spread and tissue tropism. This data will be utilised in existing SHIELD consortium screens to establish macrophage responses as tools for diagnosis or treatment of KPN infection.