Probing the Role of Protein Surface Charge in the Activation of PrfA, the Central Regulator of Listeria monocytogenes Pathogenesis

Listeria monocytogenes is a food-borne intracellular bacterial pathogen capable of causing serious human disease. L. monocytogenes survival within mammalian cells depends upon the synthesis of a number of secreted virulence factors whose expression is regulated by the transcriptional activator PrfA. PrfA becomes activated following bacterial entry into host cells where it induces the expression of gene products required for bacterial spread to adjacent cells. Activation of PrfA appears to occur via the binding of a small molecule cofactor whose identity remains unknown. Electrostatic modeling of the predicted PrfA cofactor binding pocket revealed a highly positively charged region with two lysine residues, K64 and K122, located at the edge of the pocket and another (K130) located deep within the interior. Mutational analysis of these residues indicated that K64 and K122 contribute to intracellular activation of PrfA, whereas a K130 substitution abolished protein activity. The requirement of K64 and K122 for intracellular PrfA activation could be bypassed via the introduction of the prfA G145S mutation that constitutively activates PrfA in the absence of cofactor binding. Our data indicate that the positive charge of the PrfA binding pocket contributes to intracellular activation of PrfA, presumably by facilitating binding of an anionic cofactor

Deciphering the intracellular metabolism of Listeria monocytogenes by mutant screening and modelling

Background: The human pathogen Listeria monocytogenes resides and proliferates within the cytoplasm of epithelial cells. While the virulence factors essentially contributing to this step of the infection cycle are well characterized, the set of listerial genes contributing to intracellular replication remains to be defined on a genome-wide level. Results: A comprehensive library of L. monocytogenes strain EGD knockout mutants was constructed upon insertion-duplication mutagenesis, and 1491 mutants were tested for their phenotypes in rich medium and in a Caco-2 cell culture assay. Following sequencing of the plasmid insertion site, 141 different genes required for invasion of and replication in Caco-2 cells were identified. Ten in-frame deletion mutants were constructed that confirmed the data. The genes with known functions are mainly involved in cellular processes including transport, in the intermediary metabolism of sugars, nucleotides and lipids, and in information pathways such as regulatory functions. No function could be ascribed to 18 genes, and a counterpart of eight genes is missing in the apathogenic species L. innocua. Mice infection studies revealed the in vivo requirement of IspE (Lmo0190) involved in mevalonate synthesis, and of the novel ABC transporter Lmo0135-0137 associated with cysteine transport. Based on the data of this genome-scale screening, an extreme pathway and elementary mode analysis was applied that demonstrates the critical role of glycerol and purine metabolism, of fucose utilization, and of the synthesis of glutathione, aspartate semialdehyde, serine and branched chain amino acids during intracellular replication of L. monocytogenes. Conclusion: The combination of a genetic screening and a modelling approach revealed that a series of transporters help L. monocytogenes to overcome a putative lack of nutrients within cells, and that a high metabolic flexibility contributes to the intracellular replication of this pathogen

N-Terminal Gly224–Gly411 Domain in Listeria Adhesion Protein Interacts with Host Receptor Hsp60

Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.Using a ModBase structural model, LAP was divided into 4 putative subdomains: the ALDH region contains N1 (Met(1)-Pro(223)) and N2 (Gly(224)-Gly(411)), and the ADH region contains C1 (Gly(412)-Val(648)) and C2 (Pro(649)-Val(866)). Each subdomain was cloned and overexpressed in Escherichia coli and purified. Purified subdomains were used in ligand overlay, immunofluorescence, and bead-based epithelial cell adhesion assays to analyze each domain's affinity toward Hsp60 protein or human ileocecal epithelial HCT-8 cells.The N2 subdomain exhibited the greatest affinity for Hsp60 with a K(D) of 9.50±2.6 nM. The K(D) of full-length LAP (7.2±0.5 nM) to Hsp60 was comparable to the N2 value. Microspheres (1 µm diameter) coated with N2 subdomain showed significantly (P<0.05) higher binding to HCT-8 cells than beads coated with other subdomains and this binding was inhibited when HCT-8 cells were pretreated with anti-Hsp60 antibody to specifically block epithelial Hsp60. Furthermore, HCT-8 cells pretreated with purified N2 subdomain also reduced L. monocytogenes adhesion by about 4 log confirming its involvement in interaction with epithelial cells.These data indicate that the N2 subdomain in the LAP ALDH domain is critical in initiating interaction with mammalian cell receptor Hsp60 providing insight into the molecular mechanism of pathogenesis for the development of potential anti-listerial control strategies

Constitutive Activation of PrfA Tilts the Balance of Listeria monocytogenes Fitness Towards Life within the Host versus Environmental Survival

PrfA is a key regulator of Listeria monocytogenes pathogenesis and induces the expression of multiple virulence factors within the infected host. PrfA is post-translationally regulated such that the protein becomes activated upon bacterial entry into the cell cytosol. The signal that triggers PrfA activation remains unknown, however mutations have been identified (prfA* mutations) that lock the protein into a high activity state. In this report we examine the consequences of constitutive PrfA activation on L. monocytogenes fitness both in vitro and in vivo. Whereas prfA* mutants were hyper-virulent during animal infection, the mutants were compromised for fitness in broth culture and under conditions of stress. Broth culture prfA*-associated fitness defects were alleviated when glycerol was provided as the principal carbon source; under these conditions prfA* mutants exhibited a competitive advantage over wild type strains. Glycerol and other three carbon sugars have been reported to serve as primary carbon sources for L. monocytogenes during cytosolic growth, thus prfA* mutants are metabolically-primed for replication within eukaryotic cells. These results indicate the critical need for environment-appropriate regulation of PrfA activity to enable L. monocytogenes to optimize bacterial fitness inside and outside of host cells

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence.

BACKGROUND: The polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur. RESULTS: Here, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater. CONCLUSIONS: We identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential

Glutathione activates virulence gene expression of an intracellular pathogen

Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen