The Type II Secretion System in Acinetobacter baumannii: Its Role in Pathogenesis and Translational Implications

Abstract

Acinetobacter baumannii is a Gram-negative, nosocomial pathogen that typically infects immune-compromised individuals. It is of growing concern in healthcare settings due to its ability to form biofilms, survive long periods of desiccation, and easily acquire antibiotic resistance. In an effort to better combat this pathogen, we have focused on elucidating aspects of the pathogenesis of A. baumannii. Many Gram-negative bacteria such as Vibrio cholerae and Pseudomonas aeruginosa possess a Type II Secretion System (T2SS) to secrete substrates used for colonization and survival in their environment. In our first study, we highlighted the unique organization of the T2SS genes, gsp, in A. baumannii and demonstrated that, despite this uniqueness, the T2SS is functional by analyzing a secreted lipase, LipA. Unlike the wild-type ATCC 17978 strain, both the ΔlipA and ΔgspD mutant strains were unable to grow on media where long-chain fatty acids were the only source of nutrients. This difference was also observed in a quantitative lipase assay using 4-nitrophenyl myristate as the substrate for LipA. To determine if the T2SS has a role in vivo we inoculated mice via tail vein with an equal number of the wild-type 17978 strain and a mutant strain (either the ΔlipA or ΔgspD strain). Both mutants were outcompeted by the wild-type strain indicating that the T2SS is necessary for full colonization in a bacteremia model.

We continued exploring the role of the T2SSs in pathogenesis by characterizing the metalloprotease CpaA secreted by the A. baumannii strain AB031. We verified that CpaA is a T2S substrate using an Activated Partial Thromboplastin Time (aPTT) assay, which measures the function of the intrinsic coagulation pathway, the target of CpaA. A modified aPTT assay demonstrated that CpaA targets the coagulation component factor XII (fXII). We pinpointed the site of cleavage between Pro308 and Thr309 by subjecting cleaved fXII to N-terminal sequencing by Edman degradation. As Thr309 is O-glycosylated, we tested for the necessity of O-glycosylated residues by treating fXII with deglycosylases. CpaA was unable to cleave deglycosylated fXII, but deglycosylation did not affect the function of fXII when measured by an aPTT assay. Finally, we utilized a murine bacteremia model and observed that the strain lacking CpaA was unable to compete with the wild-type strain, suggesting that it contributes to in vivo fitness possibly by preventing intravascular capture by the coagulation system. In addition to the virulence traits mentioned above, the T2SS also aids in survival in serum as the wild-type strain, 17978, withstands complement mediated killing via the alternative complement pathway significantly better than a T2SS mutant. As the T2SS contributes to in vivo survival and protection from human complement, we designed, optimized, and validated a high-throughput screen (HTS) to identify inhibitors of the T2SS in A. baumannii. By targeting the T2SS a putative inhibitor would block the secreted substrates and, thereby, their functions. By utilizing a whole-cell lipase assay, we developed a HTS with low variation among the controls and a dynamic range between the positive and negative controls, giving a z-factor of 0.65. In addition, we showed that our screen is robust, sensitive, and reproducible. Taken together, my thesis work provides much needed insight into the pathogenesis of A. baumannii by elucidating the role of the T2SS and its substrates, LipA and CpaA.