The ClpP Protease Is Required for the Stress Tolerance and Biofilm Formation in <em>Actinobacillus pleuropneumoniae</em>

<div><p>In the respiratory tract and lung tissue, a balanced physiological response is essential for <em>Actinobacillus pleuropneumoniae</em> to survive various types of challenges. ClpP, the catalytic core of the Clp proteolytic complex, is involved in various stresses response and regulation of biofilm formation in many pathogenic bacteria. To investigate the role of ClpP in the virulence of <em>A. pleuropneumoniae</em>, the <em>clpP</em> gene was deleted by homologous recombination, resulting in the mutant strain S8<em>ΔclpP</em>. The reduced growth of S8<em>ΔclpP</em> mutant at high temperatures and under several other stress conditions suggests that the ClpP protein is required for the stress tolerance of <em>A. pleuropneumoniae</em>. Interestingly, we observed that the S8<em>ΔclpP</em> mutant exhibited an increased ability to take up iron in vitro compared to the wild-type strain. We also found that the cells without ClpP displayed rough and irregular surfaces and increased cell volume relative to the wild-type strain using scanning electron microscopy (SEM). Confocal laser scanning microscopy (CLSM) revealed that the S8<em>ΔclpP</em> mutant showed decreased biofilm formation compared to the wild-type strain. We examined the transcriptional profiles of the wild type S8 and the S8<em>ΔclpP</em> mutant strains of <em>A. pleuropneumoniae</em> using RNA sequencing. Our analysis revealed that the expression of 16 genes was changed by the deletion of the <em>clpP</em> gene. The data presented in this study illustrate the important role of ClpP protease in the stress response, iron acquisition, cell morphology and biofilm formation related to <em>A. pleuropneumoniae</em> and further suggest a putative role of ClpP protease in virulence regulation.</p> </div

Polystyrene microtiter plate biofilm assay.

<p>(A) Biofilm formation of the S8, S8<i>ΔclpP</i> and S8HB strains in the wells of 96-well polystyrene microtiter plates. The plates were stained with crystal violet. (B)The quantitative determination of biofilm formation. The S8 (▪), S8<i>ΔclpP</i> (•) and S8HB (⋄) strains were grown in BHI supplemented with NAD. The optical density of the bacterial biofilm formation was monitored by OD₆₀₀ after 12, 18, 24, 30, 36 and 42 h of incubation. Points indicate the mean values, and error bars indicate standard deviations.</p

Impaired stress tolerance of the <i>A. pleuropneumoniae</i> S8<i>ΔclpP</i> mutant.

<p>Overnight cultures were inoculated into fresh medium and grown to an OD₆₀₀ value of approximately 0.6. Cells were then treated with (A) 1 mM H₂O₂ for 30 min, * p<0.01, (B) 52°C heat shock for 20 min, *p<0.01, or (C) 0.3 M KCl for 1 hour, * p<0.05. * denotes <i>P</i> values (<i>t</i> test) for comparison to S8<i>ΔclpP</i>.</p

Scanning electron microscopy.

<p>SEM of S8, S8<i>ΔclpP</i> and S8HB in the early log phase, late log phase and stationary phase were carried out. Compared to the wild-type S8 strain and the complemented S8HB strain, cells of the S8<i>ΔclpP</i> mutant show increased cell volume and rougher, more irregular surfaces. Preparation of samples was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053600#s2" target="_blank">Materials and Methods</a>.</p

The growth curves of the <i>A. pleuropneumoniae</i> in iron-restricted and iron supplemented conditions.

<p>Overnight cultures of the S8 (□), S8<i>ΔclpP</i> (○) and S8HB (▵) strains were diluted into fresh medium and then incubated in (A) BHI containing 30 µM of the iron chelator EDDHA or (B) BHI containing 30 µM of the iron chelator EDDHA and 10 µM FeSO4. Growth was monitored by OD₆₀₀ at various time points. Points indicate the mean values, and error bars indicate standard deviations.</p

Characteristics of bacterial strains, plasmids, and primers used in this study.

<p>Characteristics of bacterial strains, plasmids, and primers used in this study.</p