Characterization of a (p)ppApp Synthetase Belonging to a New Family of Polymorphic Toxin Associated with Temperate Phages.

Polymorphic toxins (PTs) are a broad family of toxins involved in interbacterial competition and pathogenesis. PTs are modular proteins that are comprised of a conserved N-terminal domain responsible for its transport, and a variable C-terminal domain bearing toxic activity. Although the mode of transport has yet to be elucidated, a new family of putative PTs containing an N-terminal MuF domain, resembling the Mu coliphage F protein, was identified in prophage genetic elements. The C-terminal toxin domains of these MuF PTs are predicted to bear nuclease, metallopeptidase, ADP-ribosyl transferase and RelA_SpoT activities. In this study, we characterized the MuF-RelA_SpoT toxin associated with the temperate phage of Streptococcus pneumoniae SPNA45. We show that the RelA_SpoT domain has (p)ppApp synthetase activity, which is bactericidal under our experimental conditions. We further determine that the two genes located downstream encode two immunity proteins, one binding to and inactivating the toxin and the other detoxifying the cell via a pppApp hydrolase activity. Finally, based on protein sequence alignments, we propose a signature for (p)ppApp synthetases that distinguishes them from (p)ppGpp synthetases.

Protein-Protein Interaction: Tandem Affinity Purification in Bacteria.

The discovery of protein-protein interaction networks can lead to the unveiling of protein complex(es) forming cellular machinerie(s) or reveal component proteins of a specific cellular pathway. Deciphering protein-protein interaction networks therefore contributes to a deeper understanding of how cells function. Here we describe the protocol to perform tandem affinity purification (TAP) in bacteria, which enables the identification of the partners of a bait protein under native conditions. This method consists in two sequential steps of affinity purification using two different tags. For that purpose, the bait protein is translationally fused to the TAP tag, which consists of a calmodulin binding peptide (CBP) and two immunoglobulin G (IgG) binding domains of Staphylococcus aureus protein A (ProtA) that are separated by the tobacco etch virus (TEV) protease cleavage site. After the first round of purification based on the binding of ProtA to IgG coated beads, TEV protease cleavage releases CBP-tagged bait-protein along with its partners for a second round of purification on calmodulin affinity resin and leaves behind protein contaminants bound to IgG. Creating the TAP-tag translational fusion at the chromosomal locus allows detection of protein interactions occurring in physiological conditions.

The iron-sulfur cluster sensor IscR is a negative regulator of Spi1 type III secretion system in Salmonella enterica.

Iron-sulfur (Fe-S)-containing proteins contribute to various biological processes, including redox reactions or regulation of gene expression. Living organisms have evolved by developing distinct biosynthetic pathways to assemble these clusters, including iron sulfur cluster (ISC) and sulfur mobilization (SUF). Salmonella enterica serovar Typhimurium is an intracellular pathogen responsible for a wide range of infections, from gastroenteritis to severe systemic diseases. Salmonella possesses all known prokaryotic systems to assemble Fe-S clusters, including ISC and SUF. Because iron starvation and oxidative stress are detrimental for Fe-S enzyme biogenesis and because such environments are often met by Salmonella during its intracellular life, we investigated the role of the ISC and SUF machineries during the course of the infection. The iscU mutant, which is predicted to have no ISC system functioning, was found to be defective for epithelial cell invasion and for mice infection, whereas the sufBC mutant, which is predicted to have no SUF system functioning, did not present any defect. Moreover, the iscU mutant was highly impaired in the expression of Salmonella pathogenicity island 1 (Spi1) type III secretion system that is essential for the first stage of Salmonella infection. The Fe-S cluster sensor IscR, a transcriptional regulator matured by the ISC machinery, was shown to bind the promoter of hilD, which encodes the master regulator of Spi1. IscR was also demonstrated to repress hilD and subsequently Spi1 gene expression, consistent with the observation that an IscR mutant is hyper-invasive in epithelial cells. Collectively, our findings indicate that the ISC machinery plays a central role in Salmonella virulence through the ability of IscR to down-regulate Spi1 gene expression. At a broader level, this model illustrates an adaptive mechanism used by bacterial pathogens to modulate their infectivity according to iron and oxygen availability.

Posttranslational maturation of the invasion acyl carrier protein of Salmonella enterica serovar Typhimurium requires an essential phosphopantetheinyl transferase of the fatty acid biosynthesis pathway.

Salmonella pathogenicity island 1 (SPI-1) carries genes required for the formation of a type 3 secretion system, which is necessary for the invasion process of Salmonella. Among the proteins encoded by SPI-1 is IacP, a homolog of acyl carrier proteins. Acyl carrier proteins are mainly involved in fatty acid biosynthesis, and they require posttranslational maturation by addition of a 4'-phosphopantetheine prosthetic group to be functional. In this study, we analyzed IacP maturation in vivo. By performing matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry analysis of intact purified proteins, we showed that IacP from Salmonella enterica serovar Typhimurium was matured by addition of 4'-phosphopantetheine to the conserved serine 38 residue. Therefore, we searched for the phosphopantetheinyl transferases in charge of IacP maturation. A bacterial two-hybrid approach revealed that IacP interacted with AcpS, an enzyme normally required for the maturation of the canonical acyl carrier protein (ACP), which is involved in fatty acid biosynthesis. The creation of a conditional acpS mutant then demonstrated that AcpS was necessary for the maturation of IacP. However, although IacP was similar to ACP and matured by using the same enzyme, IacP could not replace the essential function of ACP in fatty acid synthesis. Hence, the demonstration that IacP is matured by AcpS establishes a cross-connection between virulence and fatty acid biosynthesis pathways.

Sensing and adaptation to low pH mediated by inducible amino acid decarboxylases in Salmonella.

During the course of infection, Salmonella enterica serovar Typhimurium must successively survive the harsh acid stress of the stomach and multiply into a mild acidic compartment within macrophages. Inducible amino acid decarboxylases are known to promote adaptation to acidic environments. Three low pH inducible amino acid decarboxylases were annotated in the genome of S. Typhimurium, AdiA, CadA and SpeF, which are specific for arginine, lysine and ornithine, respectively. In this study, we characterized and compared the contributions of those enzymes in response to acidic challenges. Individual mutants as well as a strain deleted for the three genes were tested for their ability (i) to survive an extreme acid shock, (ii) to grow at mild acidic pH and (iii) to infect the mouse animal model. We showed that the lysine decarboxylase CadA had the broadest range of activity since it both had the capacity to promote survival at pH 2.3 and growth at pH 4.5. The arginine decarboxylase AdiA was the most performant in protecting S. Typhimurium from a shock at pH 2.3 and the ornithine decarboxylase SpeF conferred the best growth advantage under anaerobiosis conditions at pH 4.5. We developed a GFP-based gene reporter to monitor the pH of the environment as perceived by S. Typhimurium. Results showed that activities of the lysine and ornithine decarboxylases at mild acidic pH did modify the local surrounding of S. Typhimurium both in culture medium and in macrophages. Finally, we tested the contribution of decarboxylases to virulence and found that these enzymes were dispensable for S. Typhimurium virulence during systemic infection. In the light of this result, we examined the genomes of Salmonella spp. normally responsible of systemic infection and observed that the genes encoding these enzymes were not well conserved, supporting the idea that these enzymes may be not required during systemic infection.

Salmonella detoxifying enzymes are sufficient to cope with the host oxidative burst.

The oxidative burst produced by the NADPH oxidase (Phox) is an essential weapon used by host cells to eradicate engulfed pathogens. In Salmonella typhimurium, oxidative stress resistance has been previously proposed to be mediated by the pathogenicity island 2 type III secretion system (T3SS-2), periplasmic superoxide dismutases and cytoplasmic catalases/peroxidases. Here, we fused an OxyR-dependent promoter to the gfp to build the ahpC-gfp transcriptional fusion. This reporter was used to monitor hydrogen peroxide levels as sensed by Salmonella during the course of an infection. We showed that the expression of this fusion was under the exclusive control of reactive oxygen species produced by the host. The ahpC-gfp expression was noticeably modified in the absence of bacterial periplasmic superoxide dismutases or cytoplasmic catalases/peroxidases. Surprisingly, inactivation of the T3SS-2 had no effect on the ahpC-gfp expression. All together, these results led to a model in which Salmonella resistance relies on its arsenal of detoxifying enzymes to cope with Phox-mediated oxidative stress.

Redundant hydrogen peroxide scavengers contribute to Salmonella virulence and oxidative stress resistance.

Salmonella enterica serovar Typhimurium is an intracellular pathogen that can survive and replicate within macrophages. One of the host defense mechanisms that Salmonella encounters during infection is the production of reactive oxygen species by the phagocyte NADPH oxidase. Among them, hydrogen peroxide (H(2)O(2)) can diffuse across bacterial membranes and damage biomolecules. Genome analysis allowed us to identify five genes encoding H(2)O(2) degrading enzymes: three catalases (KatE, KatG, and KatN) and two alkyl hydroperoxide reductases (AhpC and TsaA). Inactivation of the five cognate structural genes yielded the HpxF(-) mutant, which exhibited a high sensitivity to exogenous H(2)O(2) and a severe survival defect within macrophages. When the phagocyte NADPH oxidase was inhibited, its proliferation index increased 3.7-fold. Moreover, the overexpression of katG or tsaA in the HpxF(-) background was sufficient to confer a proliferation index similar to that of the wild type in macrophages and a resistance to millimolar H(2)O(2) in rich medium. The HpxF(-) mutant also showed an attenuated virulence in a mouse model. These data indicate that Salmonella catalases and alkyl hydroperoxide reductases are required to degrade H(2)O(2) and contribute to the virulence. This enzymatic redundancy highlights the evolutionary strategies developed by bacterial pathogens to survive within hostile environments.

A bacterial pore-forming toxin forms aggregates in cells that resemble those associated with neurodegenerative diseases.

Listeria monocytogenes is a bacterial, facultative intracellular pathogen, which secretes a pore-forming toxin called listeriolysin O (LLO). LLO mediates the dissolution of the phagosomal membrane allowing L. monocytogenes to reach and grow in the host cytosolic compartment. In this study we report the localization of LLO secreted in infected cells. We described that LLO (i) forms small perinuclear aggregates, (ii) accumulates in large autophagosome-like structures and (iii) sequesters to large protein aggregates. The formation of protein aggregates required full LLO activity. Further characterization of protein aggregates indicated that they not only contained the active form of LLO but also polyubiquitinated proteins and p62, which are both common components of protein aggregates found in neurological diseases. Hence, a protein of bacterial origin could potentially follow the same fate as a toxic protein associated with neurodegenerative disease.

Use of RNA interference in Drosophila S2 cells to identify host pathways controlling compartmentalization of an intracellular pathogen.

Three genome-wide RNA interference screens were performed in Drosophila S2 cells to dissect the contribution of host processes to Listeria monocytogenes entry, vacuolar escape, and intracellular growth. Among the 116 genes identified, several host pathways previously unrecognized as playing a role in listerial pathogenesis were identified: knockdowns affecting vacuolar trafficking to and from the multivesicular body bypassed the requirement for the essential pore-forming toxin listeriolysin O in mediating escape from phagocytic vacuoles and knockdowns affecting either subunit of serine palmitoyltransferase, a key enzyme in ceramide and sphingolipid biosynthesis, enhanced the toxicity of listeriolysin O expressed in the host cell cytosol, leading to lack of appropriate toxin activity compartmentalization and host cell death. Genome-wide RNA interference screens using Drosophila S2 cells proved to be a powerful approach to dissect host-pathogen interactions.