Klebsiella pneumoniae (Kp) is a Gram-negative commensal and opportunistic bacterium of the Enterobacteriaceae family. Certain Kp strains have acquired exogenous antibiotic resistance genes or virulence genes and have become multidrug-resistant. A notable example is the carbapenem-resistant Kp (CR-Kp) strains that cause a wide range of infections such as urinary tract infection, pneumonia, bloodstream infection, and pneumonic sepsis, primarily among the elderly, immunocompromised, critically ill, and cancer patients. However, treatment options for Kp infections are limited due to the continuous evolution of multiple drug-resistant and hypervirulent variants. Hypervirulent Kp (hvKp) is currently the most common pathotype that can be differentiated from classic Kp (cKp) due to carriage of a cluster of virulence factors in a virulence plasmid or other mobile genetic elements that can be integrated into the chromosome. The threat posed by these strains has been compounded by the fact that they undergo evolution continuously, rendering them resistant to carbapenems and various other antibiotics. Evidence gathered in our laboratory and others showed that emergence of carbapenem-resistant hvKp (CR-hvKp) was due to acquisition of a pLVPK-like virulence plasmid by the carbapenem resistant strains, or plasmids containing various carbapenemase genes such as blaK_PC-2, bla_VIM, or bla_NDM-1 harbored by the hvKp strains. Therapeutic options for infections caused by CR-hvKp, which simultaneously exhibit multidrug resistance and hypervirulence, are limited, resulting in extremely high mortality among the infected patients. Thus, research on deciphering the mechanisms underlying the high mortality of hvKp strains is urgently needed to devise new approaches to prevent Kp from evading the host’s defense to enhance the effectiveness of treatment of these fatal infections.

Firstly, in an attempt to investigate the underlying mechanisms of resistance to neutrophil-mediated killing and hence expression of high-level virulence by hvKp, we tested the binding affinity of hvKp strains to various types of human cells. Our data showed that hvKp exhibited weaker binding to both lung epithelial A549 cells, intestinal Caco-2 cells when compared to the classic, non-hypervirulent strains (cKp). Consistently, hvKp strains are resistant to be engulfed or phagocyted by RAW 264.7 macrophages. Then we found that this phenotype was regulated by regulator of mucoid A (RmpA) or RmpA2 by transconjugants that have acquired a rmpA or rmpA2-bearing plasmid. These strains were found to exhibit decreased adhesion to various types of human cells, and hence higher survival rate upon exposure to neutrophil cells. Furthermore, we identified that over-production of hypermucoviscosity (HMV), but not capsular polysaccharide (CPS), contributed to the reduced binding and phagocytosis. The effect of HMV on enhancing hvKp virulence was further shown in human serum survival assays and animal experiments, indicating that HMV was largely contributed to the Kp virulence.

Secondly, we focus on the Kp-host interaction using a hvKp-induced sepsis mouse model which exhibited higher levels of bacterial burdens. To test if hvKp survives and replicates in the phagocytes, single-cell suspensions from 17ZR101-infected lungs and spleens were collected and the intracellular bacteria was counted. The result showed that hvKp could readily be engulfed and survive within phagocytes. Furthermore, scRNA-Seq revealed that hvKp infection promoted M1 polarization and neutrophil infiltration, and induced the production of inflammatory cytokines, which indicates that hvKp induced mortality was attributed to the cytokine storm. To unveil the mechanism of the regulation network of the cytokine storm, RNA-Seq was performed. We found the signal transducer and the transcription 1 (STAT1) pathway was highly activated, which further resulted in cytokine storm and extensive tissue damages. Our findings further showed that fludarabine, a STAT1 inhibitor, could rescue infected-mice form death and reduce the serum cytokine level. Importantly, we demonstrated that treatment with the commonly used non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid (ASA), which inhibits the M1 polarization process, prevented the formation of cytokine storm in the test animals, could effectively treat the fatal infections caused by hvKp. These findings highlight the value of immunosuppressive drugs in the treatment of life-threatening hvKp infections. Importantly, the combination of immunosuppressive drugs and antibiotics is more efficacious in acute hvKp infection. These findings highlight the value of immunosuppressive drugs in the treatment of acute life-threatening hvKp infections. The combination of immunosuppressive drugs and antibiotics would be an effective treatment to save the lives of patients with fatal hvKp infections, which has become common in clinical settings.

Lastly, we found that hvKp infection was accompanied by lymphopenia with reduced cell number of T cells, B cells and NK cells. This T lymphocytes reduction was associated with impaired T cell proliferation caused by T cell suppression. Lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSC) have all been associated in critically ill septic patients with persistent organ dysfunction such as acute lung injury (ALI), nosocomial infections acquisitions, and poor outcomes. The importance of emergence of myeloid-derived suppressor cells (MDSCs, CD11b⁺Gr-1⁺), that functionally suppress T-cell responses is highlighted in infectious diseases. According, we found a dramatic MDSCs expansion in 17ZR101-infected lungs and spleens, also with the M-MDSC (CD11b⁺Ly6G^-Ly6C^Hi) and PMN-MDSC (CD11b⁺Ly6G⁺Ly6C^Lo). In addition, we also detected an enhanced proportion of Treg (CD4⁺Foxp3⁺) by 17ZR101-infection. MDSCs have been reported to confer immunosuppression by various mechanisms especially in the depletion of nutrients. Then we performed untargeted metabolic analysis of lung tissues and found that the tryptophan (Trp) metabolism was the most significant affected pathway in 17ZR101-infected lungs, indicating that competed consumption of Trp in lung microenvironment resulted in the T cell proliferation inhibition. More importantly, we showed that IDO1 inhibition by 1-Methyl-D-tryptophan (1-D-MT) altered the mortality induced by 17ZR101-infection, which provided a potential target of immunotherapy in Klebsiella infection. Ex vivo assay with MDSC co-cultured with T cells suggested that T cell proliferation was inhibited by MDSC which was associated with enhanced expression of IDO1. Furthermore, by adding the L-kynurenine (L-kyn), we showed that L-kyn could inhibit T cell proliferation and induce T cell apoptosis, both of which resulted in lymphopenia. Collectively, these data indicated that Trp metabolism inhibition might be a therapeutic option in hvKp infection treatment.

Collectively, this study identified the virulence factor which contributed to hvKp survival, revealed the mechanisms underlying high mortality induced by hvKp and investigated novel therapy strategies in hvKp-infected mouse model. These data provide valuable information for future control of Kp infections in clinical settings.