lnvestigating a multidrug-resistant pathogen through the lens of T cell immunity

Klebsiella pneumoniae (Kpn) is a gram-negative, non-motile rod, endowed with a prominent sugar capsule, that is a major cause of human respiratory, urinary tract and systemic infections. Due to the rapid acquisition of antibiotic-resistance genes, the prevalence in hospitals, and the lack of a vaccine, Kpn is listed among the most dangerous multidrug-resistant (MDR) pathogens, grouped under the acronym 'ESKAPE'. In particular, carbapenem-resistant (CR)-Kpn is insensitive to virtually all available -lactams and additional classes of antibiotics, leaving scarce treatment options, and has spread worldwide. Pneumonia caused by (CR)-Kpn has an overall reported fatality rate of at least 50% and although traditionally associated to hospitalized subjects, it is occurring with increasing frequency in the general population.Recent studies have proven that upon immunization, at least in mouse models, CD4+ T cells can protect from subsequent CR-Kpn invasion, leading to the proposition that T cell-based immunotherapy could be employed to treat this infection. However, the detailed features of Kpn-specific memory T cell responses are not fully understood, nor have the antigens promoting them been identified. Additionally, we and others have shown that activation of immune responses, particularly those driven by T cells, results in rapid transcriptional adaptation of withstanding bacteria, including CR-Kpn. Some of the modulated genes appear to be involved in modulating resistance to antibiotics, consistent with the concept that targets of immunity and therapeutic molecules often overlap.Here, I propose a comprehensive study to:1) Characterize T cell responses to CR-Kpn infection in dedicated mouse modes. We will employ fate-mapping strategies and scRNA sequencing to unravel the complexity of CD4+ T cell populations primed by natural CR-Kpn infection. We will generate an ad hoc, TCR-transgenic mouse model to track CR-Kpn T cell responses in vivo. Using this tool, we will investigate key immunological aspects of CR-Kpn infection related to invasion route, inherent differences across Kpn strains, role of specific immune mediators.2) Characterize T cell responses to CR-Kpn infection in humans. Subjects that recover from disease are an excellent source of true (as opposed to cross-reactive) pathogen-specific lymphocytes. By collaborating with a clinical team, and coupling ex vivo restimulation strategies with scRNA sequencing, we will dissect the phenotypic and functional features of bona fide protective, CR-Kpn-specific T lymphocytes isolated from the blood of patients who have recovered from UTI, pulmonary, intestinal, or systemic infection. We will then utilize bioinformatic approaches to identify the immunodominant antigens recognized by the most expanded T cell clones.3) Transcriptional reprogramming or CR-Kpn and antibiotic synergy. By employing Dual-RNAseq (host/microbe) in a mous model of lung infection with CR-Kpn in the presence of distinct immunological scenarios, we will dissect the molecular adaptation programs activated by the pathogen in vivo and identify the host mediators driving them. Mining these datasets, we will then infer a series of antibiotic therapies that might synergize with, or be antagonized by, the immune response, particularly that orchestrated by memory CD4+ T cells, and test our predictions in an ex vivo model of precision cut lung slices. Finally, by using CRISPR/Cas9-mediated CR-Kpn engineering, we will causally prove the importance of identified genes in pivoting the interplay between immunity and antibiotic therapy. Our study will expand enormously our fine understanding of the events underlying immunological protection against CR-Kpn, and escape of the pathogen from leukocyte- and antibiotic-mediated killing. These studies will provide a proof of principle to explore the rational design of therapeutic approaches based on the combination of immune modulation and antibiotic treatment, advancing our capacity to tackle a nearly untreatable MDR pathogen.