Development of a Quantitative Assay Amenable for High-Throughput Screening to Target the Type II Secretion System for New Treatments against Plant-Pathogenic Bacteria

Abstract

Plant-pathogenic bacteria are the causative agents of diseases in important agricultural crops and ornamental plants. The severe economic burden of these diseases requires seeking new approaches for their control, particularly because phytopathogenic bacteria are often resistant to available treatments. The type II secretion (T2S) system is a key virulence factor used by major groups of phytopathogenic bacteria. The T2S machinery transports many hydrolytic enzymes responsible for degradation of the plant cell wall, thus enabling successful colonization and dissemination of the bacteria in the plant host. The genetic inactivation of the T2S system leads to loss of virulence, which strongly suggests that targeting the T2S could enable new treatments against plant-pathogenic bacteria. Accordingly, we have designed and optimized an assay to identify small-molecule inhibitors of the T2S system. This assay uses a double parametric output: measurement of bacterial growth and the enzymatic activity of cellulase, which is secreted via the T2S pathway in our model organism Dickeya dadantii. The assay was evaluated by screening natural extracts, culture filtrates isolated from rhizosphere bacteria, and a collection of pharmaceutically active compounds in LOPAC(1280). The calculated Z' values of 0.63, 0.63, and 0.58, respectively, strongly suggest that the assay is applicable for a high-throughput screening platform.

Jizanpeptins, Cyanobacterial Protease Inhibitors from a Symploca sp. Cyanobacterium Collected in the Red Sea

“…Chiral-phase HPLC was also used to determine the configuration of the side chain terminal 2- O -methylated glyceric acid sulfate (Mgs) in jizanpeptins B–E ( 2 – 5 ) after synthesis of methylated glyceric acid (Mga) standards following previous literature . The retention times for synthetic R - and S -Mga were compared with Mga detected in the natural product hydrolysate using an analytical Chirobiotic TAG column with a mobile phase of MeOH–NH 4 OAc.…”

“…The resulting fractions were subjected to a new preliminary biological activity screen to detect inhibition of type II secretion (T2S)-mediated virulence in pathogenic Gram-negative bacteria. This quantitative, highthroughput assay is similar in concept to that reported previously for Dickeya dadantii 16 as a model for phytopathogenic bacteria, but uses Vibrio cholerae as a model human pathogen, in which the T2S machinery and its cargo proteins are well characterized. Transcriptional activity of T2S, secretion of (or inhibition of secreted) T2S-cargo proteins (e.g., serine proteases VesA, VesB, and VesC 17 ), and bacterial growth may be assessed simultaneously.…”

“…Synthesis of R-and S-Mga was performed using a slightly modified method to that reported by Luesch et al 16 Thus, D-or L-serine (50 mg), isoamyl nitrite (141 μL), glacial acetic acid (17.2 μL), MgSO 4 (60 mg), and anhydrous MeOH (1 mL) were placed in a clear glass vial (11 mL) and heated to 110 °C (oil bath) with constant stirring for 4 h. The resulting product was cooled to rt, filtered, and dried under vacuum to yield S-/R-(racemic) or S-Mga, respectively. The observed NMR data for the crude products were in agreement with those reported previously (Figures S43−S45, Supporting Information).…”

Targeting the Type II Secretion System: Development, Optimization, and Validation of a High-Throughput Screen for the Identification of Small Molecule Inhibitors

“…In addition, our screen is designed to include high-throughput counter screens for the removal of false positives early in the triage process. The screen developed and validated by Tran et al, utilized the plant pathogen Dickeya dadantii (Tran et al, 2013 ). Similar to our screen, the authors measured OD 600 after growth to detect antibiotics and used the activity of a T2S substrate to measure the functionality of the T2SS.…”

“…Similar to our screen, the authors measured OD 600 after growth to detect antibiotics and used the activity of a T2S substrate to measure the functionality of the T2SS. As with the Moir et al, screen, the Tran et al, screen did not have counter screens developed to be utilized in the HTS process (Moir et al, 2011 ; Tran et al, 2013 ). What is apparent in all three studies is that identified compounds have to be subjected to many additional tests, including those mentioned above, before they can be classified as true T2SS inhibitors.…”