Factors involved in the survival of \(Neisseria\) \(gonorrhoeae\) in human neutrophils

Neisseria gonorrhoeae (N. gonorrhoeae), a gram-negative human pathogen that is responsible for gonorrhea, has become a critical public health concern due to its growing antibiotic resistance and limited treatment options. Since it is a human-specific pathogen, animal models have limited usage for infection studies due to species-specific differences. This is why, finding a proper model for infection studies has always been challenging. In this work, the pathogenesis of N. gonorrhoeae using already established Three Dimensional (3D) tissue models of the urothelium, endometrium, and polarized colon epithelium, which are based on a porcine Small Intestinal Submucosa (SIS) scaffold and closely mimic respective human mucosal surfaces were explored. Using these models, the dynamics of pathogen’s infection and inflammatory responses, particularly its interaction with epithelial and endothelial cell barriers, were studied. This was done upon infecting these models with different N. gonorrhoeae MS11 strain derivatives, which differ in the expressed Outer membrane porin protein B (PorB) variant. The results showed that endothelial cells strengthen tissue barrier integrity and enhance inflammatory cytokine production, particularly Interleukin (IL)-8, highlighting their importance in amplifying the host immune response. Infection with N. gonorrhoeae results in a potent neutrophil (Polymorphonuclear Leukocytes (PMNs))-driven inflammatory response. Nevertheless, viable gonococci are recovered from neutrophil-rich patient exudates, indicating that these pathogens can survive the neutrophil attack in vivo. To study the neutrophils and pathogen interactions, triple co-culture models based on T84 human colorectal carcinoma cells, human dermal fibroblast and human umbilical vein endothelial cells were developed and infected with gonococci. A perfusion based bioreactor system was used to mimic the blood flow-like conditions on the endothelial cell side, and the neutrophils transmigration to the site of infection was studied. This work could show neutrophil activation, transmigration, and recruitment to the infection site across the 3D tissue model. However, activated neutrophils showed strong non-specific attachment to the bioreactor components, which disqualified this experimental setup for further studies involving neutrophils. Considering that it is currently unclear how the bacteria survive within PMNs, identifying specific bacterial factors facilitating survival is essential to understanding this critical hostpathogen interaction. A previous screen using N. gonorrhoeae transposon library identified several bacterial factors with a potential role in the survival of gonococci upon contact with PMNs. Among the identified candidates was a cluster of genes organized in an operon (LutACB operon) which are annotated to be involved in lactate metabolism. Specific deletion of these genes in MS11 derived N2009 strain did not cause major growth defects in the culture medium but exhibited lower survival rates in neutrophils compared to the wildtype strain. As the genes involved in lactate metabolism are part of an operon, polar effects of the individual mutations were excluded. Metabolic profiling revealed disruptions in nucleotide synthesis and glutathione metabolism, which might have compromised the bacteria’s ability to withstand oxidative stress. By analyzing the adaptability of 3D models for long-term infection studies and elucidating the role of the LutACB operon in bacterial survival within neutrophils, this study provides some key insights into the host-pathogen interactions and identifies potential therapeutic targets. This study also underscores the importance of developing strategies to disrupt bacteria’s metabolic pathways and immune evasion mechanisms to address the growing antibiotic resistance threat.