Listeria monocytogenes is a facultative intracellular pathogen that escapes from phagosomes and grows in the cytosol of infected host cells. Most of the determinants that govern its intracellular life cycle are controlled by the transcription factor PrfA, including the pore-forming cytolysin listeriolysin O (LLO), two phospholipases C (PlcA and PlcB), and ActA. We constructed a strain that lacked PrfA but expressed LLO from a PrfA-independent promoter, thereby allowing the bacteria to gain access to the host cytosol. This strain did not grow efficiently in wild-type macrophages but grew normally in macrophages that lacked ATG5, a component of the autophagy LC3 conjugation system. This strain colocalized more with the autophagy marker LC3 (42% ؎ 7%) at 2 h postinfection, which constituted a 5-fold increase over the colocalization exhibited by the wild-type strain (8% ؎ 6%). While mutants lacking the PrfA-dependent virulence factor PlcA, PlcB, or ActA grew normally, a double mutant lacking both PlcA and ActA failed to grow in wild-type macrophages and colocalized more with LC3 (38% ؎ 5%). Coexpression of LLO and PlcA in a PrfA-negative strain was sufficient to restore intracellular growth and decrease the colocalization of the bacteria with LC3. In a cell-free assay, purified PlcA protein blocked LC3 lipidation, a key step in early autophagosome biogenesis, presumably by preventing the formation of phosphatidylinositol 3-phosphate (PI3P). The results of this study showed that avoidance of autophagy by L. monocytogenes primarily involves PlcA and ActA and that either one of these factors must be present for L. monocytogenes growth in macrophages.

Cholesterol-dependent cytolysins (CDCs) represent a family of homologous pore-forming proteins secreted by many Gram-positive bacterial pathogens. CDCs mediate membrane binding partly through a conserved C-terminal undecapeptide, which contains a single cysteine residue. While mutational changes to other residues in the undecapeptide typically have severe effects, mutation of the cysteine residue to alanine has minor effects on overall protein function. Thus, the role of this highly conserved reactive cysteine residue remains largely unknown. We report here that the CDC listeriolysin O (LLO), secreted by the facultative intracellular pathogen , was posttranslationally modified by S-glutathionylation at this conserved cysteine residue and that either endogenously synthesized or exogenously added glutathione was sufficient to form this modification. When recapitulated with purified protein, this modification completely ablated the activity of LLO, and this inhibitory effect was fully reversible by treatment with reducing agents. A cysteine-to-alanine mutation in LLO rendered the protein completely resistant to inactivation by S-glutathionylation, and a mutant expressing this mutation retained full hemolytic activity. A mutant strain of expressing the cysteine-to-alanine variant of LLO was able to infect and replicate within bone marrow-derived macrophages indistinguishably from the wild type, yet it was attenuated 4- to 6-fold in a competitive murine infection model This study suggests that S-glutathionylation may represent a mechanism by which CDC-family proteins are posttranslationally modified and regulated and help explain an evolutionary pressure to retain the highly conserved undecapeptide cysteine.

The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of Listeria monocytogenes.

The association of membrane proteins with a single transmembrane a-helix (TMH) has been shown to play a critical role in numerous cellular processes.[1] For example, TMH dimerization of the ErbB family of receptor tyrosine kinases (RTKs) results in trans-phosphorylation and subsequent activation of the downstream pathways of growth factor signalling.[2] A series of naturally occurring, single amino acid mutations in the TMH domain of these RTKs have been linked to a number of diseases, presumably because these mutations perturb TMH dimerization propensities.[1] For example, the dimer promoting mutation V664E in the TMH domain of ErbB2 (HER2) has been associated with an increased risk of cancer. [3,4] This physiological importance is reflected in the high number of drugs that target membrane proteins (ca. 50 %) relative to their distribution in the proteome (ca. 30 %). [5] Although significant progress has been made in our mechanistic understanding of protein association in membranes, [6][7][8][9][10] it remains difficult to predict the oligomerization state of a TMH and rationalize the (patho)physiological consequences of a mutation within a membrane protein. While the association of soluble proteins is primarily driven by the hydrophobic effect, protein-protein interaction within membranes is thought to rely on weak molecular interactions such as sidechain hydrogen bonding and van der Waal's packing.[6] In order to gain a better understanding of these energetic factors and ultimately develop novel inhibitors of TMH association, a simple and efficient assay is highly desirable for quantifying TMH association both in vitro (detergent micelles and liposomes) and in living cells.Biarsenical dyes, such as 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH), are an intriguing tool for monitoring protein-protein interaction in membranes.In their EDT (ethane-1,2-dithiol)-protected forms, these dyes (e.g., FlAsH-EDT 2 ) are essentially nonfluorescent. However, thiol-arsenic ligand exchange with a tC motif elicits a strong fluorescence emission.[11] These fluorophores have been applied to a number of soluble proteins to report their folding and stability, and even subtle conformational changes. [11][12][13][14][15][16][17][18][19][20] A recent development in this area has resulted in a technique known as "bipartite tC display", in which the biarsenical dyes are used to monitor the dimerization of water-soluble proteins.[18] Herein, we describe the development of a FlAsH-tC assay for quantifying the association of membrane-embedded proteins. While the commonly used methods, such as SDS-PAGE and analytical ultracentrifugation (AUC) are limited to micellar systems, [21] the FlAsH-tC assay allows facile evaluation of protein dimerization in both micelles and lipid bilayers. Importantly, the FlAsH fluorescence reports TMH dimerization in an orientation-specific manner (vide infra), which is advantageous over the previously reported, FRET-based assays. [22] As a model system, we first synthesised a pair of polyleucine (pL)-b...

Broad‐range, phosphatidylcholine‐preferring phospholipase C (PC‐PLCLM) from Listeria monocytogenes is one of the critical virulence factors mediating the dissolution of vacuolar membranes so that the intracellular pathogen can escape into the cytosol and spread to neighboring cells. We have expressed the enzyme in E. coli and characterized its kinetics with a variety of substrates. The amino acid sequence of PC‐PLCLM is highly homologous to Bacillus cereus PC‐PLCBC. Both are metalloenzymes with identical residues coordinating to the Zn2+ ligands. Intriguingly, the PC‐PLCLM exhibits an acidic pH optimum (pH5~6) compared to the well‐studied PC‐PLCBC which is more efficient at basic pH values. This is significant as PC‐PLCLM activity is needed to aid in Listeria escape from acidified vacuolar pH. The optimal efficiency at acidic pH results from dramatically reduced Km values but moderate changes in kcat at lower pH. In addition, unlike PLCBC, PC‐PLCLM can cleave sphingomyelin. Mechanistic details for the acidic pH optimum and substrate specificity are probed with mutagenesis and a combination of biochemical and biophysical approaches. This study is supported by the National Institutes of Health GM60418.

Listeriolysin O (LLO), as one of the major virulence factors of intracellular pathogen Listera monocytogenes, assists in the escape of the bacterium from the phagosome by specifically forming oligomeric transmembrane β‐barrels on the target membrane. Its unique acidic pH optimum lessens the toxicity towards the host cell. Cholesterol is claimed to be the membrane receptor of LLO with the C‐terminal domain 4 (D4) involved in the binding and recognition. In contrast, domains 1 to 3 (D1‐3) are critical for monomer aggregation on the membrane. To better investigate these two distinct interactions with the membrane, we have separately expressed domains D4 and D1‐3 and compared their behavior to full‐length LLO using a variety of biophysical approaches (TEM, FCS, NMR). Full‐length LLO is lytic only towards cholesterol‐containing membranes and at acidic pH. D4 binds to membranes regardless of cholesterol or pH, presumably due to the hydrophobicity of its tip (ECTGLAWEWWR). However, this domain can be stabilized in a well‐folded form in DPC and SDS micelles; these have sufficient resolution in 15N‐1H HSQC experiments for some structural work. D1‐3 is nonlytic and soluble, but it aggregates significantly with the aggregation enhanced at acidic pH. These results are used to propose a modified model for the action of LLO on target membranes. These studies were supported by the NIH (GM60418).