Bacterial antimicrobial resistance is becoming a severe burden to human health and the medical system. Combating this challenge involves understanding drug resistance enzymes and virulence factors. This project aims to characterize the drug resistance enzyme TetX, bacteria cell surface virulence factor pseudaminic acid (Pse5Ac7Ac). Cell surface carbohydrate pseudaminic acid (Pse) is involved in virulence in bacteria, and it was found on the surface of several multi-drug resistance bacteria. Pseudaminic acid is not commercially available, and the primary source is through chemical synthesis, which is time-consuming, low quantity, and expensive. Limited access to pseudaminic acid set back the study of biological significance and the potential target of novel antimicrobial agents. This study aims to establish an enzymatic synthesis method to produce an acceptable quantity of pseudaminic acid and reduce the cost.

In this study, six recombinant pseudaminic acid synthesis enzymes from Acinetobacter baumannii were cloned and purified for enzymatic synthesis. One-pot synthesis was performed to produce pseudaminic acid (Pse) and nucleotide-activated pseudaminic acid (CMP-Pse) at the milligram level. In addition, functional analysis was performed to examine the biological significance of pseudaminic acid. We knocked out the production of pseudaminic acid in Acinetobacter baumannii and performed animal experiments and in-vitro experiments, and we discovered that the Pse plays a significant role in the survival of Acinetobacter in the host, thus increasing the virulence. The Pse acts by binding to a complement factor H, subsequently inhibiting the activation of the complement system. As a result, the complement-killing mechanism of bacteria is inhibited. The presence of pse inside bacteria helps it to escape from host killing mechanism, thus increasing the virulence.

Tigecycline is a last-resort antibiotic to fight multiple drug-resistant (MDR) bacterial infections. However, the emergence of flavin adenine dinucleotide (FAD)- dependent monooxygenase TetX has led to resistance to tigecycline. In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. We provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes, which is currently unavailable. Our findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist the inactivation of the TetX variants.