UC Riverside

The rapid emergence of resistant bacteria is arising and causing a global health crisis. Antibiotic resistance is a natural phenomenon. Bacteria can be intrinsically resistant to certain antibiotics but can also acquire resistance to antibiotics via mutations in chromosomal genes and by horizontal genes. The success of antibiotics might only have been temporary. Our knowledge of the diverse mechanisms involved has notably increased in recent years. It provides us greater understanding to aid the discovery and development of new agents. We now expect to find new approaches to circumvent or neutralize existing resistance mechanisms.

In the thesis, we briefly overview the current antibiotic resistance threat of the society. To delve into how pathogens fighting back, three major antibiotic resistance molecular mechanisms were described.

(1) Prevention of access to targets; it includes the lack of a susceptible target of a specific antibiotic, the down-regulation of porins or by the replacement of prions with more-selective channels, and the overexpression of efflux pumps.

(2) Target protection; this includes the changes to the target structure to prevent efficient antibiotic binding, the acquisition of a gene homologous to the original target to gaining new resistance, and the modification of the target.

(3) Directly modification of the antibiotic molecules; it contains the hydrolysis of antibiotics and the transfer of chemical groups.

Based on the understanding of resistance mechanisms, researchers have identified 19 alternatives-to-antibiotics approaches for consideration. A few approaches under extensive studies in this list were illustrated here. For instance, peptidomimetic antimicrobials can cause membrane lipid bending to complete dissolution; aminoglycosides and derivatives interact with 16S RNA and affect translational fidelity; nanoparticles possess a unique ability to cross into bacterial cell with strengthened antibacterial activity; probiotics as live microorganisms confer a health benefit to the host organism; and quorum sensing (QS) quenching acts as a down-regulation of virulent factors expression.

We specifically aimed at the fifth approach – QS quenching. It is based on the fact that bacterial QS system regulates the expression of non-essential functions - virulence factors by sensing their own population density. Manipulating QS system instead of affecting growth rate as antibiotics may decrease the development of resistance. One approach to interfering with QS system is by the usage of QS inhibitors. QS inhibitors include QS molecule analogues and other diverse biochemical molecule structures. As the competitive inhibitors to the real communication signals autoinducers (AIs), QS inhibitors interfere with QS communication, thus disrupting biofilm formation and inhibiting the expression of virulence factors. Technically, QS inhibitors does not directly cause cell death; therefore applying less selective. However, recently it has been discovered that pathogens tend to evolve resistance by increasing efflux of quorum quenching (QQ) agents in some cases.

The other approach to disrupting QS is the use of QQ enzymes abolishing the biological activity of AIs. The existence of quorum quenching enzymes in the quorum sensing microbes can attenuate their quorum sensing, leading to blocking unnecessary gene expression and pathogenic phenotypes. QQ enzymes have been identified in quorum sensing and non-quorum sensing microbes, including lactonase, decarboxylase, acylase, deaminase and oxidoreductase. The first report of degradation AIs was lactonase AiiA isolated from Bacillus sp. 240B. Later we used AiiA for our studies as a positive QQ enzyme control because of its broad-spectrum AHL-degrading ability. QQ microbes have been identified in a range of Gram-negative and Gram-positive microorganisms. Yet for therapeutic purposes, prokaryotic enzymes are very likely to generate adverse immune responses. It would be desirable to have a therapeutic enzyme that is as close as possible to the native human protein with high catalytic efficiency, minimal immunogenicity, and soluble expression with low production cost. Currently, there is no such enzyme meeting all these requirements.

Human paraoxonases (huPONs) have been discovered to have AHL-lactonase like activity, hydrolyzing lactones of various modifications and carbon chain lengths. In fact, according to recently studies, huPONs play an important role in innate immunity, inflammatory response, and protection against oxidative stress; these factors are associated with the body’s response to infectious diseases. Especially for huPON2, it modulates stress response of endothelial cells to oxidized phospholipids and a bacterial quorum-sensing molecule. However, mammalian paraoxonases are cell-membrane or high-density-lipoprotein (HDL)-associated enzymes. They are difficult to express in soluble forms. And when heterogeneously expressed, the yield is considerably low.

Thus, we intent to enhance huPON2 performance by improving its solubility, yield and activity. Through replacing the protruding hydrophobic helices of huPON2 with degenerate short peptide linkers, and from the constructed linker library of limited design diversity, we isolated human PON2 variants exhibiting a high level of soluble expression. The engineered huPON2 have both lactone hydrolysis activities toward a spectrum of QS molecules found in clinically important pathogens and are biologically functional in P. aeruginosa swimming, swarming motility and biofilm formation tests. And then, we tried to generate random mutations to the soluble expressed huPON2s to improve its catalytic activity.