Identification of Nanaomycin A and Its Analogs as Functional Inhibitors of the Type IX Secretion System in Porphyromonas gingivalis Using a Novel Screening Method

Academic Background

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium recognized as one of the primary pathogens in chronic periodontitis. Beyond periodontal diseases, P. gingivalis is also closely associated with various systemic conditions such as diabetes, rheumatoid arthritis, dementia, and preterm birth. Therefore, reducing the virulence of P. gingivalis is a critical strategy for treating these diseases.

P. gingivalis secretes a series of highly hydrolytic proteases, known as gingipains, through its Type IX Secretion System (T9SS). These proteases enable the bacterium to obtain peptides from the environment as an energy source. Although T9SS-related proteins have been extensively studied, no specific inhibitors targeting T9SS have been identified to date. Thus, developing a method to screen for T9SS inhibitors is of great significance for understanding the functional mechanisms of T9SS and for developing new therapeutic agents for periodontal diseases.

Source of the Paper

This paper was co-authored by Yuko Sasaki, Takehiro Matsuo, Yoshihiro Watanabe, Masato Iwatsuki, Yuki Inahashi, Satoshi Nishida, Mariko Naito, and Mikio Shoji. The research team is affiliated with Nagasaki University, Kitasato University, and Teikyo University. The paper was published online on November 22, 2024, in The Journal of Antibiotics, with the DOI 10.1038/s41429-024-00790-8.

Research Process and Results

1. Research Background and Objectives

The objective of this study was to develop a novel screening method for identifying functional inhibitors of T9SS. To achieve this, the research team focused on a minimal liquid medium called MC Medium (Milk Casein Medium), which uses milk casein as the sole protein source. By observing the turbidity changes of P. gingivalis in MC medium, the team aimed to identify compounds that could inhibit T9SS function.

2. Characterization of MC Medium

The research team first validated the properties of MC medium. They found that the wild-type strain of P. gingivalis, ATCC 33277, could increase the turbidity of MC medium without bacterial growth. Further experiments demonstrated that Iodoacetamide (IAM) and Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP) significantly inhibited the increase in turbidity. IAM is an alkylating agent that inhibits gingipain activity, while CCCP is a protonophore that disrupts the proton motive force required for T9SS.

Additionally, the team discovered that gingipain-null mutants and T9SS-deficient mutants were unable to increase the turbidity of MC medium, indicating that the turbidity change in MC medium depends on gingipain activity and T9SS function.

3. Screening for T9SS Inhibitors

Based on the properties of MC medium, the research team developed a high-throughput screening method to identify T9SS inhibitors. They used a natural compound library containing 603 compounds (Ōmura Natural Compound Library) and conducted the screening using 96-well plates. The screening criteria were that the compounds should inhibit the increase in turbidity in MC medium without affecting bacterial growth in BHI medium.

Through the screening, the team identified two compounds, OM-173αA and OM-173βA, which significantly inhibited the increase in turbidity in MC medium without affecting bacterial growth in BHI medium. Further experiments revealed that these compounds are analogs of Nanaomycin A.

4. Mechanism of Action of Nanaomycin A and Its Analogs

The research team further investigated the mechanism of action of Nanaomycin A and its analogs. They found that these compounds inhibited the degradation of casein proteins in MC medium but did not directly inhibit gingipain activity. This suggests that Nanaomycin A and its analogs may inhibit the secretion of gingipains by affecting T9SS function.

Moreover, the team discovered that Nanaomycin A and its analogs significantly reduced gingipain activity in MC medium, particularly in Rgp (arginine-specific protease) activity. This indicates that these compounds may prevent the accumulation of gingipains in MC medium by inhibiting T9SS function.

5. Experimental Validation and Data Analysis

To validate the mechanism of action of these compounds, the research team conducted multiple experiments. They analyzed the degradation of casein proteins in MC medium using SDS-PAGE and found that Nanaomycin A and its analogs significantly inhibited casein degradation. Additionally, they verified the effects of these compounds on gingipain activity through enzymatic assays.

Data analysis showed that the inhibitory effects of Nanaomycin A and its analogs in MC medium were dose-dependent, with an IC50 value of approximately 5 µM. These results further support the potential of these compounds as functional inhibitors of T9SS.

Conclusions and Significance

The study concludes that MC medium can serve as an effective screening tool for identifying functional inhibitors of T9SS. Using this method, the research team successfully identified Nanaomycin A and its analogs as potential T9SS inhibitors. These compounds not only provide new tools for studying the functional mechanisms of T9SS but also offer potential candidates for developing new therapeutic agents for periodontal diseases.

Highlights of the Study

1. Development of a Novel Screening Method: This study is the first to utilize the properties of MC medium to develop a high-throughput screening method for T9SS inhibitors, providing a new tool for studying T9SS function.

2. Discovery of Nanaomycin A and Its Analogs: The research team successfully identified Nanaomycin A and its analogs as functional inhibitors of T9SS, which have potential clinical applications.

3. Elucidation of the Mechanism of Action: Through a series of experiments, the team revealed that Nanaomycin A and its analogs inhibit the secretion of gingipains by affecting T9SS function, offering new insights into the study of T9SS.

Additional Valuable Information

The study also found that CCCP inhibited the growth of P. gingivalis in BHI medium but exhibited a stronger inhibitory effect in MC medium. This suggests that CCCP may inhibit bacterial growth by affecting T9SS function rather than solely inhibiting gingipain activity.

Furthermore, the team observed that different strains of P. gingivalis exhibited varying degrees of turbidity changes in MC medium, which may be related to differences in gingipain expression levels among strains. This finding provides new clues for further research into the virulence mechanisms of P. gingivalis.

Summary

This study successfully identified Nanaomycin A and its analogs as functional inhibitors of T9SS by developing a novel screening method. These findings not only provide new tools for studying the functional mechanisms of T9SS but also offer potential candidates for developing new therapeutic agents for periodontal diseases. Future research will further explore the mechanisms of action of these compounds and evaluate their potential for clinical applications.