Inhibitor of Chromosome Segregation in Pseudomonas aeruginosa from Fungal Extracts

Abstract

Chromosome segregation is an essential cellular process that has the potential to yield numerous targets for drug development. This pathway is presently underutilized partially due to the difficulties in the development of robust reporter assays suitable for high throughput screening. In bacteria, chromosome segregation is mediated by two partially redundant systems, condensins and ParABS. Based on the synthetic lethality of the two systems, we developed an assay suitable for screening and then screened a library of fungal extracts for potential inhibitors of the ParABS pathway, as judged by their enhanced activity on condensin-deficient cells. We found such activity in extracts of Humicola sp. Fractionation of the extract led to the discovery of four new analogues of sterigmatocystin, one of which, 4-hydroxy-sterigmatocystin (4HS), displayed antibacterial activity. 4HS induced the phenotype typical for parAB mutants including defects in chromosome segregation and cell division. Specifically, bacteria exposed to 4HS produced anucleate cells and were impaired in the assembly of the FtsZ ring. Moreover, 4HS binds to purified ParB in a ParS-modulated manner and inhibits its ParS-dependent CTPase activity. The data describe a small molecule inhibitor of ParB and expand the known spectrum of activities of sterigmatocystin to include bacterial chromosome segregation.

Figure 1

Screening strategy. (A) Diagram of chromosome and cell duplication in P. aeruginosa. Replication of the daughter chromosome often begins before the mother chromosomes are fully replicated. The mother chromosome is replicated in the middle of the cell, while the daughters replicate at cell quarters. These locations are also the place where the divisome assembles, once initiated by the formation of FtsZ rings. Condensins help define these locations while also creating a scaffold for the chromosome. ParAB facilitates the segregation of the replication origins. (B) Schematic rationale of the screen. Inhibitors of ParAB are expected to selectively inhibit the growth of condensin mutants. (C) Growth inhibition of PΔ3-Pore (WT), PΔ3-Pore parB (parB), and PΔ3-Pore smc mksB (smc mksB) cells by the most active hit compound (n = 3; ± SD).

Figure 2

Sterigmatocystin analogues isolated from Humicola spp including 4HS (1), methoxysterigmatocystin (5), sterigmatocystin (6), and deoxysterigmatocystin (7). MIC and, in parentheses, IC₅₀ (μg/mL) for PΔ3-Pore smc mksB are shown below each compound.

Figure 3

Anucleate cell formation induced by 4HS. (A) Overlayed fluorescence micrographs of PΔ3-Pore (WT) cells exposed or not to 4HS at 0.5× MIC. Cells were incubated with or without 4HS overnight, fixed with ethanol, washed, supplemented with DAPI and SyproOrange to stain the DNA (red) and protein (black), and observed. Size bar, 2 μm. (B) The population of anucleate cells (among >300 cells) following the exposure to 4HS. p-values were determined using a two-tailed t-test; error bars are SD (n ≥ 3).

Figure 4

Cell division defects induced by 4HS or mutational inactivation of ParAB. (A) Localization of FtsZ-mCherry in PΔ3-Pore (WT) and PΔ3-Pore parB cells. Size bar, 2 μm. (B) The proportions of each type of cells (n ≥ 154) observed in the experiment from panel A. (C) Cumulative cell length distribution in exponential cells exposed to 4HS for the indicated times or mutationally devoid of ParB or ParAB (n > 1000).

Figure 5

Growth inhibition by 4HS (n = 2; ± SD) of cells that harbor the empty vector (pUCP22) or plasmids that overproduce MksB (pMksB) or SMC (pSMC).

Figure 6

4HS binding and inhibition of ParB in vitro. (A) Microscale thermophoresis analysis of 4HS binding to 100 nM ParB in the absence or presence of 1 μM ParS. The data were fit to a 1:1 interaction model. (B) The rate of CTP hydrolysis (n = 2; ± SD) by 1 μM ParB in the presence of 1 μM ParS normalized to ParB monomers fits the Michaelis–Menten equation. (C). CTPase rate of 1 μM ParB in the absence of DNA or in the presence of 1 μM ParS or randomized ParS (rnd) DNA and the indicated amount of 4HS (n = 2; ± SD).

Figure 7

Predicted binding site of 4HS in the P. aeruginosa ParAB complex. (A) The structures of ParA (cyan) and ParB (magenta) were predicted and relaxed using AlphaFold-Multimer and docked to 4HS (CPK representation) using the Schrodinger software suite. The structure was further aligned to a DNA-bound ParB (blue) from C. crescentus(47) (PDB ID: 6S6H). (B) 4HS in its top-ranked docked pose forms two hydrogen bonds with ParAB.