Proteomic Analyses of aListeria monocytogenesMutant Lacking σ^BIdentify New Components of the σ^BRegulon and Highlight a Role for σ^Bin the Utilization of Glycerol

Abstract

In Listeria monocytogenes the alternative sigma factor B plays important roles in both virulence and stress tolerance. In this study a proteomic approach was used to define components of the B regulon in L. monocytogenes 10403S (serotype 1/2a). Using two-dimensional gel electrophoresis and the recently developed isobaric tags for relative and absolute quantitation technique, the protein expression profiles of the wild type and an isogenic ⌬sigB deletion strain were compared. Overall, this study identified 38 proteins whose expression was B dependent; 17 of these proteins were found to require the presence of B for full expression, while 21 were expressed at a higher level in the ⌬sigB mutant background. The data obtained with the two proteomic approaches showed limited overlap (four proteins were identified by both methods), a finding that highlights the complementarity of the two technologies. Overall, the proteomic data reaffirmed a role for Listeria monocytogenes is a gram-positive facultative intracellular pathogen associated with a life-threatening disease in humans (43). Infections occur in immunocompromised individuals following ingestion of contaminated food. The success of L. monocytogenes as a human pathogen is determined in part by the presence of specialized virulence genes that allow it to multiply and spread within the host and in part by its ability to persist in harsh environmental conditions. It is now clear that there is a strong link between the virulence potential of L. monocytogenes and its ability to tolerate stress. Several studies have identified genes that play important roles in stress tolerance and virulence. For example, genes involved in osmotic stress tolerance (11, 37), acid tolerance (40), oxidative stress tolerance (17), and bile salt tolerance (36) have all been implicated in the virulence of L. monocytogenes. The link between stress tolerance and virulence is further underlined by the fact that several dedicated virulence genes are stress inducible (38,39).In all bacteria an effective response to stress requires rapid adaptation at the transcriptional level to changing environmental conditions. L. monocytogenes regulates the transcription of many genes with stress-related functions by employing an alternative sigma factor, B . Mutants lacking B display increased sensitivity to a range of stresses, such as osmotic pressure (4, 21, 42), acid (19,42,46), heat, ethanol, and oxidative stress (18), cold (4), and high hydrostatic pressure (44). Deletion of sigB also decreases the ability of L. monocytogenes to survive in the presence of bacteriocins and antibiotics (5).More recently, roles for B in regulating the expression of dedicated virulence genes have been described. A mutant lacking B expresses the internalin gene, inlA, at a reduced level, and this correlates with a defect in its ability to invade human epithelial cells (25). Recently, B was also shown to be solely responsible for transcription of the inlC2 and inlD internalin genes, while it coregulates expression of the inlA...

Refinement of the Listeria monocytogenes σB regulon through quantitative proteomic analysis

“…One study only used 2-DE (Abram et al, 2008b), and although the other study used iTRAQ as well as 2-DE, it used L. monocytogenes cells grown to stationary phase in 0.5 M salt for iTRAQ analysis, which is different from the growth conditions used to generate the microarray data that were used for the meta-analysis detailed below. The study that used both iTRAQ and 2-DE (Abram et al, 2008a) identified 17 proteins as positively regulated by s B , compared to 36 proteins identified here; 14 of these 36 proteins had also been reported as positively regulated by s B in this previous study. The 2-DE study by Abram et al (2008b), on the other hand, found 10 proteins to be positively regulated by s B ; 7 of these proteins were also found among the 36 proteins we identified here as positively regulated by s B .…”

“…Proteins were isolated from stationary phase L. monocytogenes cells from 25 ml of culture using the method of Abram et al (2008a) with slight modifications as previously described (Mujahid et al, 2013). Protein concentrations were determined using a noninterfering protein assay kit (Calbiochem).…”

“…Along with transcriptional analyses, proteomic studies have also been used to define the s B regulon in L. monocytogenes 10403S. Using non-gel based quantitative proteomics and two-dimensional gel electrophoresis (2-DE), Abram et al (2008a) identified 17 proteins that showed higher expression levels in the parent strain than the DsigB strain, using bacteria grown to stationary phase in Brain Heart Infusion (BHI) medium with or without osmotic stress (0.5 M NaCl). Additionally, a 2-DE study of bacteria grown to exponential or stationary phase in chemically defined media with or without 0.5 M NaCl (Abram et al, 2008b) identified 10 proteins that showed higher levels in the parent strain as compared to the DsigB mutant.…”

“…Overall, the studies detailed above show that s B in L. monocytogenes is responsible for positive regulation of a large regulon with .100 genes, with a considerable number of these genes directly regulated by s B , as supported by identification of s B consensus promoters upstream of many of these genes (Abram et al, 2008a;Oliver et al, 2009Oliver et al, , 2010Raengpradub et al, 2008); however, a comprehensive analysis that formally integrates data on the s B regulon from multiple studies has been missing so far. Increasing evidence further suggests that s B also makes important contributions to gene regulation in L. monocytogenes via mechanisms other than direct regulation of a gene or operon through s B -dependent transcription from an upstream promoter, including, but not limited to, regulation of non-coding RNAs (ncRNAs), which can regulate gene expression at both the transcriptional and post-transcriptional stage (Mellin & Cossart, 2012;Nielsen et al, 2008;Oliver et al, 2009;Toledo-Arana et al, 2009).…”

Development and optimization of an EGFP-based reporter for measuring the general stress response inListeria monocytogenes

“…A variety of stress and virulence-related phenotypes are associated with loss of the sigB gene, indicating that s B plays an important role both during infections and under stressful conditions (reviewed by O'Byrne, 2008). 5 After the full genome sequence of L. monocytogenes became available 6 a number of studies identified components of the s B regulon by looking at genes differentially expressed in L. monocytogenes wild-type and corresponding sigB mutants using proteomic approaches 7,8 or gene microarray technology. [9][10][11][12] The mechanism responsible for sensing stress that leads to the activation of s B has not been fully characterized in L. monocytogenes.…”