Subcellular Localization of Extracytoplasmic Proteins in Monoderm Bacteria: Rational Secretomics-Based Strategy for Genomic and Proteomic Analyses

Abstract

Genome-scale prediction of subcellular localization (SCL) is not only useful for inferring protein function but also for supporting proteomic data. In line with the secretome concept, a rational and original analytical strategy mimicking the secretion steps that determine ultimate SCL was developed for Gram-positive (monoderm) bacteria. Based on the biology of protein secretion, a flowchart and decision trees were designed considering (i) membrane targeting, (ii) protein secretion systems, (iii) membrane retention, and (iv) cell-wall retention by domains or post-translocational modifications, as well as (v) incorporation to cell-surface supramolecular structures. Using Listeria monocytogenes as a case study, results were compared with known data set from SCL predictors and experimental proteomics. While in good agreement with experimental extracytoplasmic fractions, the secretomics-based method outperforms other genomic analyses, which were simply not intended to be as inclusive. Compared to all other localization predictors, this method does not only supply a static snapshot of protein SCL but also offers the full picture of the secretion process dynamics: (i) the protein routing is detailed, (ii) the number of distinct SCL and protein categories is comprehensive, (iii) the description of protein type and topology is provided, (iv) the SCL is unambiguously differentiated from the protein category, and (v) the multiple SCL and protein category are fully considered. In that sense, the secretomics-based method is much more than a SCL predictor. Besides a major step forward in genomics and proteomics of protein secretion, the secretomics-based method appears as a strategy of choice to generate in silico hypotheses for experimental testing.

Phenotypes Associated with the Essential Diadenylate Cyclase CdaA and Its Potential Regulator CdaR in the Human Pathogen Listeria monocytogenes

“…The TM domain is attached to the diadenylate cyclase (DAC) domain, which is surrounded by two coiled-coil (CC) motifs. CdaR is predicted to contain a signal peptide (SP) and four YbbR domains of unknown function (24,25,26).…”

“…3B). Moreover, truncated CdaR variants lacking the signal peptide (24,25,26) showed interactions with the full-length NCdaR (pKT25 derivative) and C-CdaR (p25-N derivative) proteins. This observation might indicate that the CdaR variants can interact with each other via their YbbR domains even though they are not properly inserted into the membrane and thus are cytoplasmic.…”

“…It is predicted to contain an N-terminal signal peptide (aa 1 to 33) and four YbbR domains of unknown function (Fig. 1B) (24,25,26). This particular genetic context of the cdaA gene suggests that CdaA may be involved in cell envelope biosynthesis.…”

Surface adhesins and exopolymers of selected foodborne pathogens

“…Cell surface proteins are the major adhesion factors contributing to surface colonization in L. monocytogenes (Renier et al, 2011). According to the most recent proteogenomic analyses based on the secretome concept (Desvaux et al, 2009;Renier et al, 2012), 58 secreted proteins were predicted to be located in the cell wall, of which 43 correspond to LPXTG proteins covalently attached to the cell wall and 15 correspond to GW (five), WXL (four), LysM (five) and PGBD1 (one) proteins attached to the cell wall by noncovalent interactions. Furthermore, numerous proteins are predicted to be located at the cytoplasmic membrane, namely 74 lipoproteins and 686 integrated membrane proteins (IMPs), in addition to cell surface supramolecular protein structures, namely the pseudo-pilus and flagellum.…”

Listeria monocytogenesencodes a functional ESX-1 secretion system whose expression is detrimental toin vivoinfection

“…1). Interestingly, Lmo0062 was recently predicted to be secreted, 38 therefore investigating its capacity to be secreted in an ESX-1-dependent manner could widen the spectrum of ESX-1 substrates to proteins that do not belong to the WXG100 family.…”