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Salmonella spp. can cause animal and human salmonellosis. In this study, we established a simple method to detect all Salmonella species by amplifying a specific region within the flgE gene encoding the flagellar hook protein. Our preliminary sequence analysis among flagella-associated genes of Salmonella revealed that although Salmonella Gallinarum and Salmonella Pullorum are lacking flagella, they did have flagella-associated genes, including flgE. To investigate in detail, a comparative flgE sequence analysis was conducted using different bacterial strains including flagellated and non-flagellated Salmonella as well as non-Salmonella strains. Two unique regions (481-529 bp and 721-775 bp of the reference sequence) within the flgE open reading frame were found to be highly conserved and specific to all Salmonella species. Next, we designed a pair of PCR primers (flgE-UP and flgE-LO) targeting the above two regions, and performed a flgE-tailored PCR using as template DNA prepared from a total of 76 bacterial strains (31 flagellated Salmonella strains, 26 non-flagellated Salmonella strains, and 19 other non-Salmonella bacteria strains). Results showed that specific positive bands with expected size were obtained from all Salmonella (including flagellated and non-flagellated Salmonella) strains, while no specific product was generated from non-Salmonella bacterial strains. PCR products from the positive bands were confirmed by DNA sequencing. The minimum detection amount for genomic DNA and bacteria cells reached 18.3 pg/μL and 100 colony-forming unit (CFU) per PCR reaction, respectively. Using the flgE-PCR method to detect Salmonella in artificially contaminated milk samples, as low as 1 CFU/mL Salmonella was detectable after an 8-h pre-culture. Meanwhile, the flgE-tailored PCR method was applied to evaluate 247 clinical samples infected with Salmonella from different chicken breeding farms. The detection results indicated that flgE-PCR could be used to specifically detect Salmonella in concordance with the traditional bacterial culture-based detection method. It is worthwhile noticed that identification results using flgE-tailored PCR should be completed within less than 1 day, expanding the result of much faster than the standard method, which took more than 5 days. Overall, the flgE-tailored PCR method can specifically detect flagellated and non-flagellated Salmonella and can serve as a powerful tool for rapid, simple, and sensitive detection of Salmonella species. KEY POINTS : • Targeting flgE gene for all Salmonella spp. found. • The established PCR assay is used to specifically detect all Salmonella spp. • The PCR method is applied to detect clinical Salmonella spp. samples within less than 1 day.

Many Gram-negative bacterial pathogens interact with mammalian cells by using type III secretion systems (T3SS) to inject virulence proteins into host cells. A subset of these injected protein ‘effectors’ are enzymes that inhibit the function of host proteins by catalyzing the addition of unusual post-translational modifications. The E. coli and Citrobacter rodentium NleB effectors, as well as the Salmonella enterica SseK effectors are glycosyltransferases that modify host protein substrates with N-acetyl glucosamine (GlcNAc) on arginine residues. This post-translational modification disrupts the normal functioning of host immune response proteins. T3SS effectors are thought to be inactive within the bacterium and fold into their active conformations after they are injected, due to the activity of chaperones that keep the effectors in a structural state permissive for secretion. While performing mass spectrometry experiments to identify glycosylation substrates of NleB orthologs, we unexpectedly observed that the bacterial glutathione synthetase (GshB) is glycosylated by NleB on arginine residue R256. NleB-mediated glycosylation of GshB resulted in enhanced GshB activity, leading to an increase in glutathione production, and promoted C. rodentium survival in oxidative stress conditions. These data represent, to our knowledge, the first intra-bacterial activity for a T3SS effector and show that arginine-GlcNAcylation, once thought to be restricted to host cell compartments, also plays an important role in regulating bacterial physiology.

Whether type III secretion system (T3SS) effector proteins encoded by Gram-negative bacterial pathogens have intra-bacterial activities is an important and emerging area of investigation. Gram-negative bacteria interact with their mammalian hosts by using secretion systems to inject virulence proteins directly into infected host cells. Many of these injected protein effectors are enzymes that modify the structure and inhibit the function of mammalian proteins. The underlying dogma is that T3SS effectors are inactive until they are injected into host cells, where they then fold into their active conformations. We previously observed that the T3SS effectors NleB and SseK1 glycosylate Citrobacter rodentium and Salmonella enterica proteins, respectively, leading to enhanced resistance to environmental stress. Here, we sought to extend these studies to determine whether the T3SS effector protease NleC is also active within C. rodentium. To do this, we expressed the best-characterized mammalian substrate of NleC, the NF-κB p65 subunit in C. rodentium and monitored its proteolytic cleavage as a function of NleC activity. Intra-bacterial p65 cleavage was strictly dependent upon NleC. A p65 mutant lacking the known CE cleavage motif was resistant to NleC. Thus, we conclude that, in addition to NleB, NleC is also enzymatically active within C. rodentium.

Type III secretion system effector proteins have primarily been characterized for their interactions with host cell proteins and their ability to disrupt host signaling pathways. We are testing the hypothesis that some effectors are active within the bacterium, where they modulate bacterial signal transduction and physiology. We previously determined that the Citrobacter rodentium effector NleB possesses an intra-bacterial glycosyltransferase activity that increases glutathione synthetase activity to protect the bacterium from oxidative stress. Here we investigated the potential intra-bacterial activities of NleB orthologs in Salmonella enterica and found that SseK1 and SseK3 mediate resistance to methylglyoxal. SseK1 glycosylates specific arginine residues on four proteins involved in methylglyoxal detoxification, namely GloA (R9), GloB (R190), GloC (R160), and YajL (R149). SseK1-mediated Arg-glycosylation of these four proteins significantly enhances their catalytic activity, thus providing another important example of the intra-bacterial activities of type three secretion system effector proteins. These data are also the first demonstration that a Salmonella T3SS effector is active within the bacterium.

NleB/SseK effectors are arginine-GlcNAc-transferases expressed by enteric bacterial pathogens that modify host cell proteins to disrupt signaling pathways. While the conserved Citrobacter rodentium NleB and E. coli NleB1 proteins display a broad selectivity towards host proteins, Salmonella enterica SseK1, SseK2, and SseK3 have a narrowed protein substrate selectivity. Here, by combining computational and biophysical experiments, we demonstrate that the broad protein substrate selectivity of NleB relies on Tyr284NleB/NleB1, a second-shell residue contiguous to the catalytic machinery. Tyr284NleB/NleB1 is important in coupling protein substrate binding to catalysis. This is exemplified by S286YSseK1 and N302YSseK2 mutants, which become active towards FADD and DR3 death domains, respectively, and whose kinetic properties match those of enterohemorrhagic E. coli NleB1. The integration of these mutants into S. enterica increases S. enterica survival in macrophages, suggesting that better enzymatic kinetic parameters lead to enhanced virulence. Our findings provide insights into how these enzymes finely tune arginine-glycosylation and, in turn, bacterial virulence. In addition, our data show how promiscuous glycosyltransferases preferentially glycosylate specific protein substrates., The NleB and SseK glycosyltransferases glycosylate arginine residues of mammalian proteins with different substrate specificities. We uncover that these differences rely on a particular second-shell residue contiguous to the catalytic machinery.

The enteropathogenic and enterohemorrhagic Escherichia coli NleB proteins as well as the Salmonella enterica SseK proteins are type III secretion system effectors that function as glycosyltransferase enzymes to post-translationally modify host substrates on arginine residues. This modification is unusual because it occurs on the guanidinium groups of arginines, which are poor nucleophiles, and is distinct from the activity of the mammalian O-linked N-acetylglucosaminyltransferase. We conducted high-throughput screening assays to identify small molecules that inhibit NleB/SseK activity. Two compounds, 100066N and 102644N, both significantly inhibited NleB1, SseK1, and SseK2 activities. Addition of these compounds to cultured mammalian cells was sufficient to inhibit NleB1 glycosylation of the tumor necrosis factor receptor type 1-associated DEATH domain protein. These compounds were also capable of inhibiting Salmonella enterica strain ATCC 14028 replication in mouse macrophage-like cells. Neither inhibitor was significantly toxic to mammalian cells, nor showed in vitro cross-reactivity with the mammalian O-linked N-acetylglucosaminyltransferase. These compounds or derivatives generated from medicinal chemistry refinements may have utility as a potential alternative therapeutic strategy to antibiotics or as reagents to further the study of bacterial glycosyltransferases.

Bacteria deliver virulence proteins termed 'effectors' to counteract host innate immunity. Protein-protein interactions within the host cell ultimately subvert the generation of an inflammatory response to the infecting pathogen. Here we briefly describe a subset of T3SS effectors produced by enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), Citrobacter rodentium, and Salmonella enterica that inhibit innate immune pathways. These effectors are interesting for structural and mechanistic reasons, as well as for their potential utility in being engineered to treat human autoimmune disorders associated with perturbations in NF-κB signaling.

Bacteria deliver virulence proteins termed ‘effectors’ to counteract host innate immunity. Protein-protein interactions within the host cell ultimately subvert the generation of an inflammatory response to the infecting pathogen. Here we briefly describe a subset of T3SS effectors produced by enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), Citrobacter rodentium, and Salmonella enterica that inhibit innate immune pathways. These effectors are interesting for structural and mechanistic reasons, as well as for their potential utility in being engineered to treat human autoimmune disorders associated with perturbations in NF-κB signaling.

15 pags, 5 figs, The bacterial effector proteins SseK and NleB glycosylate host proteins on arginine residues, leading to reduced NF-κB-dependent responses to infection. Salmonella SseK1 and SseK2 are E. coli NleB1 orthologs that behave as NleB1-like GTs, although they differ in protein substrate specificity. Here we report that these enzymes are retaining glycosyltransferases composed of a helix-loop-helix (HLH) domain, a lid domain, and a catalytic domain. A conserved HEN motif (His-Glu-Asn) in the active site is important for enzyme catalysis and bacterial virulence. We observe differences between SseK1 and SseK2 in interactions with substrates and identify substrate residues that are critical for enzyme recognition. Long Molecular Dynamics simulations suggest that the HLH domain determines substrate specificity and the lid-domain regulates the opening of the active site. Overall, our data suggest a front-face Si mechanism, explain differences in activities among these effectors, and have implications for future drug development against enteric pathogens., This work was supported by Grants from the National Research Foundation of Korea (NRF) funded by the Korean government (MEST) (NRF- 2016R1A2B2013305, 2016R1A5A1010764, 2014R1A4A1008625, 2017M3A9F6029755 and 2017R1A2B3006704), the Strategic Initiative for Microbiomes in Agriculture and Food funded by Ministry of Agriculture, Food and Rural Affairs (918012-4) and Brain Korea 21 PLUS Project for Medical Science. This work was also supported by Grants AI093913 and AI127973 from the National Institutes of Health (to P.R.H.). We also thank ARAID and MEC (CTQ2013-44367-C2-2-P, BFU2016-75633-P to R.H-G.), and the DGA (group number E34_R17) for financial support. S.M. acknowledges a postgraduate studentship from the School of Pharmacy of the University of East Anglia. J.A. and S W. acknowledge funding from BBSRC through a research grant (BB/P010660/1) and a DTP PhD studentship, respectively.

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-d-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium. Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and C. rodentium NleB blocked TNF-mediated NF-κB pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg197 and Arg200, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.

Many Gram-negative bacterial pathogens use a syringe-like apparatus called a type III secretion system to inject virulence factors into host cells. Some of these effectors are enzymes that modify host proteins to subvert their normal functions. NleB is a glycosyltransferase that modifies host proteins with N-acetyl-D-glucosamine to inhibit antibacterial and inflammatory host responses. NleB is conserved among the attaching/effacing pathogens enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium. Moreover, Salmonella enterica strains encode up to three NleB orthologs named SseK1, SseK2, and SseK3. However, there are conflicting reports regarding the activities and host protein targets among the NleB/SseK orthologs. Therefore, here we performed in vitro glycosylation assays and cell culture experiments to compare the activities and substrate specificities of these effectors. SseK1, SseK3, EHEC NleB1, EPEC NleB1, and C. rodentium NleB blocked TNF-mediated NF-B pathway activation, whereas SseK2 and NleB2 did not. C. rodentium NleB, EHEC NleB1, and SseK1 glycosylated host GAPDH. C. rodentium NleB, EHEC NleB1, EPEC NleB1, and SseK2 glycosylated the FADD (Fas-associated death domain protein). SseK3 and NleB2 were not active against either substrate. We also found that EHEC NleB1 glycosylated two GAPDH arginine residues, Arg and Arg, and that these two residues were essential for GAPDH-mediated activation of TNF receptor-associated factor 2 ubiquitination. These results provide evidence that members of this highly conserved family of bacterial virulence effectors target different host protein substrates and exhibit distinct cellular modes of action to suppress host responses.

Vitamin B 6 is an essential cofactor for a large number of enzymes in both prokaryotes and eukaryotes. In this study, we characterized the pyridoxal 5′-phosphate (PLP) biosynthesis pathway in Streptococcus pneumoniae . Our results revealed that S. pneumoniae possesses a de novo vitamin B 6 biosynthesis pathway encoded by the pdxST genes. Purified PdxS functionally displayed as PLP synthase, whereas PdxT exhibited glutaminase activity in vitro . Deletion of pdxS , but not pdxT , resulted in a vitamin B 6 auxotrophic mutant. The defective growth of the Δ pdxS mutant in a vitamin B 6 -depleted medium could be chemically restored in the presence of the B 6 vitamers at optimal concentrations. By analyzing PdxS expression levels, we demonstrated that the expression of pdxS was repressed by PLP and activated by a transcription factor, PdxR. A pneumococcal Δ pdxR mutant also exhibited as a vitamin B 6 auxotroph. In addition, we found that disruption of the vitamin B 6 biosynthesis pathway in S. pneumoniae caused a significant attenuation in a chinchilla middle ear infection model and a minor attenuation in a mouse pneumonia model, indicating that the impact of vitamin B 6 synthesis on virulence depends upon the bacterial infection niche.

Vibrio anguillarumutilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like otherVibriospecies,V. anguillarumcontains the geneqrr1, which encodes the ancestral quorum regulatory RNA Qrr1, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Qrr sRNAs were identified. All four sRNAs were positively regulated by σ54and the σ54-dependent response regulator VanO, and showed a redundant activity. The Qrr sRNAs, together with the RNA chaperone Hfq, destabilizedvanTmRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all fourqrrgenes peaked at high cell density, and exogenously addedN-acylhomoserine lactone molecules induced expression of theqrrgenes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Qrr sRNAs and stabilizedvanTmRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Qrr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

International audience; Soupene et al. [J. Bacteriol. (2003) 185 5611-5626] made the unexpected observation that the presence of a mutation, in the gene for the N-acetylglucosamine repressor, nagC, increased the growth rate of Escherichia coli MG1655 on galactose, an unrelated sugar. We have found that NagC, binds to a single, high-affinity site overlapping the promoter of galP (galactose permease) gene and that expression of galP is repressed by a combination of NagC, GalR and GalS. In addition to the previously identified galOE operator, other gal operators further upstream are required for full repression. GalS has a specific role, as it binds with higher affinity to one of the upstream operators but its effect in vivo is only observed in the presence of GalR. Regulation of galP by three specific repressors, NagC, GalR and GalS is unusual in that it involves multiple, specific regulators from two different areas of metabolism. This novel regulation seems to be particular for E. coli and its nearest neighbour, Shigella. Other bacteria with galP orthologues, although retaining the metK-galP gene order, do not have the NagC site. Although quantitative effects were strain specific, nagC mutations increased the growth rate on galactose of all E. coli strains tested.

The Mlc and NagC transcriptional repressors bind to similar 23-bp operators. The sequences are weakly palindromic, with just four positions totally conserved. There is no cross regulation observed between the repressors in vivo, but there are no obvious bases which could be responsible for operator site discrimination. To investigate the basis for operator recognition and to try to understand what differentiates NagC sites from Mlc sites, we have undertaken mutagenesis experiments to convert ptsG from a gene regulated by Mlc into a gene regulated by NagC. There are two Mlc operators upstream of ptsG , and to switch ptsG to the NagC regulon, it was necessary to change two different characteristics of both operators. Firstly, we replaced the AT base pair at position +/−11 from the center of symmetry of the operators with a GC base pair. Secondly, we changed the sequence of the CG base pairs in the central region of the operator (positions −4 to +4 around the center of symmetry). Our results show that changes at either of these locations are sufficient to lose regulation by Mlc but that both types of changes in both operators are necessary to convert ptsG to a gene regulated by NagC. In addition, these experiments confirmed that two operators are necessary for regulation by NagC. We also show that regulation of ptsG by Mlc involves some cooperative binding of Mlc to the two operators.