Comparative proteome analysis of secretory proteins from pathogenic and nonpathogenicListeriaspecies

Abstract

Extracellular proteins of bacterial pathogens play a crucial role in the infection of the host. Here we present the first comprehensive validation of the secretory subproteome of the Gram positive pathogen Listeria monocytogenes using predictive bioinformatic and experimental proteomic approaches. The previous original signal peptide (SP) prediction (Glaser et al., Science 2001, 294, 849-852) has been greatly improved by an in-depth analysis using seven different bioinformatic tools. Subsequent careful classification of the resulting data gives a probability dependent annotation of 121 putatively secreted proteins of which 45 are novel. Complementary proteomic analysis using both two-dimensional gel electrophoresis/matrix assisted laser desorption/ionization mass spectrometry and high performance liquid chromatography/electrospray ionization-mass spectrometry has identified 105 proteins in the culture supernatant of L. monocytogenes. Among these, we were able to detect all the currently known virulence factors with an SP showing the importance of this subproteome and demonstrating the reliability of the techniques used. The comparison between the L. monocytogenes wildtype and the nonpathogenic species Listeria innocua was performed to reveal proteins probably involved in pathogenicity and/or the adaptation to their respective lifestyles. In addition to the eight known virulence factors, all of which have no orthologous genes in L. innocua, eight additional proteins have been identified that exhibit the typical key feature defining the known listerial virulence factors. Further significant differences between the two species are evident in the group of cell wall and secretory proteins that warrant further study. Our investigation clearly demonstrates that the major difference between the pathogenic and nonpathogenic species, noted in the comparative genome analysis, manifests itself strongest in the secretome.

Listeria monocytogenesSurface Proteins: from Genome Predictions to Function

“…The autolysins P60 and MurA, which contain two and four LysM domains, respectively, are present in purified cell wall fractions (34). Lmo1303 and Lmo2522, which contain one and two LysM domains, respectively, are detected in supernatant fractions, indicating that these proteins can translocate across the plasma membrane (179). One LysM domain is also found in Lmo1941, a protein with a transmembrane hydrophobic domain that is detected in the L. monocytogenes membrane fraction (193).…”

“…Proteomic studies identified proteins that have no predicted signal sequence, and are assumed to have cytoplasmic functions, in the L. monocytogenes cell wall or supernatant fractions (160,179). Among them are glycolytic enzymes, chaperones, and heat shock proteins, as well as proteins involved in detoxification and adaptation to atypical conditions, nucleic acid metabolism, transcription, and translation.…”

“…Through extensive in silico analysis of the genome of L. monocytogenes EGDe, Trost et al (179) were able to predict a total of 525 sequences coding for proteins carrying a signal peptide, which corresponds to 18.4% of the total number of protein-coding genes. Among them, the "surface proteins" discussed in this review are those carrying motifs enabling strong or weak interactions with at least one component of the bacterial envelope (Fig.…”

Differential Secretomics of Streptococcus pyogenes Reveals a Novel Peroxide Regulator (PerR)-regulated Extracellular Virulence Factor Mitogen Factor3 (MF3)

“…The proteomic approach has been successfully used to address a global view of bacterially physiological issues (15)(16)(17)(18)(19)(20). Here, we compared S. pyogenes secretome samples from the wild-type strain and the perR deficient mutant by using gel-based proteomics analysis for discovery of novel PerR-regulated extracellular virulence factors.…”

Two-dimensional gel electrophoresis in bacterial proteomics

“…Secretome analysis of various pathogenic bacteria, including Aeromonas hydrophila, L. monocytogenes, H. pylori, B. anthracis, B. subtilis, S. aureus and Pseudomonas aeruginosa, has not only identified known virulence factors, but has also revealed previously uncharacterized secreted proteins that were involved in pathogenesis Nouwens et al, 2002;Tjalsma et al, 2004;Trost et al, 2005;Chitlaru et al, 2006;Sibbald et al, 2006;Yu et al, 2007). For example, in S. aureus, the function of the diacylglycerol transferase (Lgt) virulence factor was revealed by comparing the exoproteomes of wild type and Lgt mutants with impaired virulence (Sibbald et al, 2006).…”