Role of (p)ppGpp in Viability and Biofilm Formation of Actinobacillus pleuropneumoniae S8.

Gang Li,Chunlai Wang,Yanhe Zhang,Janine T Bossé,Fang Xie,Paul R Langford,Willem Van Schaik

doi:10.1371/journal.pone.0141501

Abstract

Actinobacillus pleuropneumoniae is a Gram-negative bacterium and the cause of porcine pleuropneumonia. When the bacterium encounters nutritional starvation, the relA-dependent (p)ppGpp-mediated stringent response is activated. The modified nucleotides guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and guanosine 5’-triphosphate 3’-diphosphate (pppGpp) are known to be signaling molecules in other prokaryotes. Here, to investigate the role of (p)ppGpp in A. pleuropneumoniae, we created a mutant A. pleuropneumoniae strain, S8ΔrelA, which lacks the (p)ppGpp-synthesizing enzyme RelA, and investigated its phenotype in vitro. S8ΔrelA did not survive after stationary phase (starvation condition) and grew exclusively as non-extended cells. Compared to the wild-type (WT) strain, the S8ΔrelA mutant had an increased ability to form a biofilm. Transcriptional profiles of early stationary phase cultures revealed that a total of 405 bacterial genes were differentially expressed (including 380 up-regulated and 25 down-regulated genes) in S8ΔrelA as compared with the WT strain. Most of the up-regulated genes are involved in ribosomal structure and biogenesis, amino acid transport and metabolism, translation cell wall/membrane/envelope biogenesis. The data indicate that (p)ppGpp coordinates the growth, viability, morphology, biofilm formation and metabolic ability of A. pleuropneumoniae in starvation conditions. Furthermore, S8ΔrelA could not use certain sugars nor produce urease which has been associated with the virulence of A. pleuropneumoniae, suggesting that (p)ppGpp may directly or indirectly affect the pathogenesis of A. pleuropneumoniae during the infection process. In summary, (p)ppGpp signaling represents an essential component of the regulatory network governing stress adaptation and virulence in A. pleuropneumoniae.

Highlights

Actinobacillus pleuropneumoniae is a non-motile Gram-negative bacterium causing porcine pleuropneumonia, a highly contagious respiratory disease that is transmitted through aerosolsPLOS ONE | DOI:10.1371/journal.pone.0141501 October 28, 2015(p)ppGpp Affect Actinobacillus pleuropneumoniae S8
Total RNA was isolated from A. pleuropneumoniae S8 and S8HB strains grown to early stationary phase (12 h) in Tryptic Soy Broth (TSB) broth for analysis of relA expression
In a previous study by Lone et al [34], genes involved in the stringent response were shown to be up-regulated in a malT mutant of A pleuropneumoniae grown in porcine bronchoalviolar doi:10.1371/journal.pone.0141501.g009

Summary

Introduction

Actinobacillus pleuropneumoniae is a non-motile Gram-negative bacterium causing porcine pleuropneumonia, a highly contagious respiratory disease that is transmitted through aerosols. The stringent response is a broadly conserved bacterial stress response that controls adaptation to nutrient deprivation, and is activated by a number of different starvation and stress signals. This response is used by bacteria to determine resource allocation for either reproductive or cell maintenance functions [2]. A. pleuropneumoniae can adhere to cells of the lower respiratory tract in a process involving different adhesins and probably biofilm formation [13]. In this site, A. pleuropneumoniae causes tissue damage leading to clinical disease and mortality [13]. The results suggest that (p)ppGpp directly or indirectly affects the pathogenesis of A. pleuropneumoniae

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Discussion