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

Mutations Blocking Side Chain Assembly, Polymerization, or Transport of a Wzy-Dependent Streptococcus pneumoniae Capsule Are Lethal in the Absence of Suppressor Mutations and Can Affect Polymer Transfer to the Cell Wall

Extracellular polysaccharides of many bacteria are synthesized by the Wzy polymerase-dependent mechanism, where long-chain polymers are assembled from undecaprenyl-phosphate-linked repeat units on the outer face of the cytoplasmic membrane. In gram-positive bacteria, Wzy-dependent capsules remain largely cell associated via membrane and peptidoglycan linkages. Like many Wzy-dependent capsules, the Streptococcus pneumoniae serotype 2 capsule is branched. In this study, we found that deletions of cps2K, cps2J, or cps2H, which encode a UDP-glucose dehydrogenase necessary for side chain synthesis, the putative Wzx transporter (flippase), and the putative Wzy polymerase, respectively, were obtained only in the presence of suppressor mutations. Most of the suppressor mutations were in cps2E, which encodes the initiating glycosyltransferase for capsule synthesis. The cps2K mutants containing the suppressor mutations produced low levels of high-molecular-weight polymer that was detected only in membrane fractions. cps2K-repaired mutants exhibited only modest increases in capsule production due to the effect of the secondary mutation, but capsule was detectable in both membrane and cell wall fractions. Lethality of the cps2K, cps2J, and cps2H mutations was likely due to sequestration of undecaprenyl-phosphate in the capsule pathway and either preclusion of its turnover for utilization in essential pathways or destabilization of the membrane due to an accumulation of lipid-linked intermediates. The results demonstrate that proper polymer assembly requires not only a functional transporter and polymerase but also complete repeat units. A central role for the initiating glycosyltransferase in controlling capsule synthesis is also suggested

Identification of a Peptide-Pheromone that Enhances <i>Listeria monocytogenes</i> Escape from Host Cell Vacuoles

<div><p><i>Listeria monocytogenes</i> is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following <i>L</i>. <i>monocytogenes</i> entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for <i>L</i>. <i>monocytogenes</i> secretion of a small peptide pheromone, pPplA, which enhances the escape of <i>L</i>. <i>monocytogenes</i> from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to <i>L</i>. <i>monocytogenes</i> its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol.</p></div

pPplA enhances bacterial aggregation in broth culture.

<p><b>(A)</b> Image of bacterial aggregation observed between the wild-type <i>L</i>. <i>monocytogenes</i> 10403S strain versus a <i>prfA</i>* mutant when bacterial cultures grown in BHI are left statically overnight at room-temperature (RT). <b>(B)</b> Measurement of the rate of bacterial aggregation in BHI. The optical-density at 600nm was monitored at the indicated time points for 1 mL of an overnight culture initially grown in BHI with shaking at 37°C then left statically at RT, where bacterial aggregation is measured as the decrease in the optical-density of the culture supernatant as the bacterial aggregate out of solution. <b>(C)</b> Measurement of bacterial aggregation of the indicated mutant strains resuspended in 1 mL of spent media derived from overnight stationary phase cultures either containing the pPplA peptide (G72stop) or lacking it (Δ<i>pplA</i>). The ability of the pPplA containing media (G72stop) to restore bacterial aggregation indicates the presence of a secreted substance (potentially pPplA) that enhances bacterial aggregation in broth culture. <b>(D)</b> Measurement of bacterial aggregation as done in panel C, except strains were resuspended in 1 mL of BHI spent media derived from <i>E</i>. <i>coli</i> containing the complementation vector construct expressing the N-terminal 72 amino acids of PplA as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.g002" target="_blank">Fig. 2A</a> or the empty vector. The ability of spent media derived from an <i>E</i>.<i>coli</i> strain containing the first 72 amino acids of pPplA supports the secretion of a PplA N-terminus derived peptide that enhances bacterial aggregation. <b>(E)</b> Assessment of bacterial aggregation in a strain containing three amino acid substitutions within the predicted peptide sequence (referred to as <i>prfA</i>*<i>pplA</i>^<i>m</i>). Reduced aggregation of <i>prfA</i>*<i>pplA</i>^<i>m</i> indicates the importance of these amino acids within the pPplA pheromone. <b>(F)</b> Bacterial aggregation of two oligopeptide transport mutants, a <i>prfA</i>* Δ<i>ctaP</i> compared to a <i>prfA</i>*-<i>oppA</i> insertion mutant. A <i>prfA</i>* Δ<i>ctaP</i> is impaired for bacterial aggregation, indicating a possible link between the pPplA peptide and import of the peptide through the CtaP transport system. For panels (B), (C) and (D), data is representative of at least three independent experiments.</p

Oligonucleotides used in this study.

<p>^<i>a</i>Italicized letters indicate restriction enzymes used in making constructs. Primer pairs used for construction of an in-frame <i>pplA</i> deletion mutant were <i>pplA-S</i>oeA-<i>XbaI</i> and <i>pplA-S</i>oeB to amplify the upstream flanking region and <i>pplA</i>-SoeC and <i>pplA</i>-SoeD-<i>EcoRI</i> to amplify the downstream flanking region, both <i>pplA</i>-SoeB and <i>pplA</i>-SoeC contain internal <i>KpnI</i> sites for insertion of the <i>ermB</i> gene (similar combination of primer pairs were used for construction of the respective <i>pplA</i>-G72 stop codon mutant and the <i>eep</i> deletion mutant, but <i>eep</i>-SoeA contained a <i>PstI</i> site and <i>eep</i>-SoeD contained a <i>SacI</i> site). <i>oppA</i> gene-disruption primers contained a <i>KpnI</i> site, pIMK2C’F a <i>NcoI</i> site, pIMK2C’R a <i>XmaI</i> site, <i>pplA</i>His-F a <i>KpnI</i> site, and <i>pplA</i>His-R an <i>XmaI</i> site.</p><p>^<i>b</i>Letters in bold indicate the premature stop codon engineered at amino acid position G72 in the <i>pplA</i> coding sequence.</p><p>Oligonucleotides used in this study.</p

Model of <i>L</i>. <i>monocytogenes</i> pPplA signaling within the host vacuole.

<p>Modeling of the predicted <i>L</i>. <i>monocytogenes</i> pPlpA signaling pathway involved in enhancing vacuole escape from the host cell vacuoles in non-professional phagocytic cells. In wild-type <i>L</i>. <i>monocytogenes</i>, <i>pplA</i> encodes a lipoprotein (PplA) with a peptide pheromone (pPplA) located within the N terminal secretion signal peptide (shown in green). The signal sequence of prePplA is processed by signal peptidase II (SPII) and the released signal peptide is further cleaved by the protease Eep releasing the pPplA pheromone, while the PplA protein becomes lipid modified and associated with the membrane or secreted. Upon entry of wild-type <i>L</i>. <i>monocytogenes</i> into non-professional phagocytic host cell, the confined space of the vacuole leads to import of the secreted pPplA pheromone through the CtaP peptide transporter. pPplA accumulation in the bacterial cytoplasm stimulates a signaling cascade that results in the production of an unknown factor (X) that contributes to vacuole lysis. Factor X may function by helping to stabilize the LLO generated membrane pore, facilitating eventual vacuole membrane dissolution as well as the influx of mammalian cytosol components that may promote PrfA activation and the expression of gene products required for intracellular growth and cell-to-cell spread. In the absence of pPplA (Δ<i>pplA</i> strains), bacterial escape is delayed until sufficient LLO and phospholipase accumulate to disrupt the vacuole membrane in the absence of factor X function. For strains containing constitutively activated PrfA*, the substantially increased secretion of LLO and the phospholipases is sufficient to disrupt the vacuole membrane in the absence of factor X.</p

Constitutive activation of <i>prfA</i>* rescues virulence defects associated with loss of pPplA pheromone.

<p><b>(A)</b> Intracellular growth of the <i>prfA</i>* mutant, the wild-type strain and a <i>prfA</i>*Δ<i>pplA</i> mutant in PtK2 epithelial cells. Gentamicin was added one hour p.i. to kill extracellular bacteria, coverslips were removed at the indicated time points, host cells were lysed and intracellular bacteria were enumerated. <b>(B)</b> Swiss Webster female mice were intravenously inoculated with 2 x 10⁴ CFUs through the tail vein, and the livers and spleens were harvested 48 hours post-infection (p.i.) and homogenized to determine bacterial burdens. The Δ<i>pplA</i> single mutant data is from data presented in Figs. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.g004" target="_blank">4</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.g005" target="_blank">5</a> and is meant to represent a point of reference. The addition of a <i>prfA</i>* mutation to the Δ<i>pplA</i> mutant is able to completely restore any virulence defects associated with loss of <i>pplA</i>, suggesting a link between the pPplA pheromone function and PrfA activation.</p

<i>L</i>. <i>monocytogenes</i> secretes a PrfA-induced lipoprotein that shares homology with <i>Enterococcus faecalis</i> Cad and its N-terminal encoded peptide-pheromone.

<p><b>(A)</b> Protein alignment of <i>E</i>. <i>faecalis</i> Cad to <i>L</i>. <i>monocytogenes</i> Lmo2637 (PplA) using ClustalW2 software program (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). The signal sequence region encoding the cAD1 peptide-pheromone and the predicted pPlpA peptide are respectively highlighted in green and red for <i>Ef</i> (<i>E</i>. <i>faecalis</i>) or blue for <i>Lm</i> (<i>L</i>. <i>monocytogenes</i>), and the lipoprotein portion of the proteins is highlighted in tan. <b>(B)</b> Gene organization of the <i>pplA</i> coding region. The presence of a putative transcriptional terminator downstream of <i>pplA</i> is indicated by the circle with stem.</p

Loss of <i>pplA</i> pheromone does not delay intracellular growth or vacuolar escape in professional phagocytic cells.

<p>Intracellular growth of the wild-type strain compared to the Δ<i>pplA</i> mutant in (A) J774 macrophage-like cells and (B) murine bone-marrow derived macrophages (BMMØ) using an MOI of 01:1. Loss of <i>pplA</i> did not impair intracellular growth inside professional phagocytic cells. Data shown is representative of three independent experiments.</p

Secreted protein spots present in both wild-type 10403S and the <i>pplA</i>-G72_STOP mutant but absent in the in-frame <i>pplA</i> deletion mutant.

<p>^<i>a</i>EGD-e designations.</p><p>^<i>b</i>SecA2 secreted proteins identified in Lenz <i>et al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.ref088" target="_blank">88</a>] and in Renier <i>et</i>. <i>al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.ref089" target="_blank">89</a>] or possibly secreted by the SecA2 system as these proteins have been identified as non-classically secreted proteins in Bendtsen <i>et al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.ref087" target="_blank">87</a>] or by using SecretomeP 2.0 Server program (<a href="http://www.cbs.dtu.dk/services/SecretomeP/" target="_blank">http://www.cbs.dtu.dk/services/SecretomeP/</a>); SecA secreted proteins identified in Port and Freitag [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.ref038" target="_blank">38</a>] and/or Baumgartner <i>et</i>. <i>al</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004707#ppat.1004707.ref103" target="_blank">103</a>], or predicted by SignalP 4.1 Server program (<a href="http://www.cbs.dtu.dk/services/SignalP/" target="_blank">http://www.cbs.dtu.dk/services/SignalP/</a>).</p><p>^<i>c</i>Unique peptide matches.</p><p>Secreted protein spots present in both wild-type 10403S and the <i>pplA</i>-G72_STOP mutant but absent in the in-frame <i>pplA</i> deletion mutant.</p