QseBC controls flagellar motility, fimbrial hemagglutination and intracellular virulence in fish pathogen Edwardsiella tarda

The inter-kingdom communication with the mammalian hosts mediated by autoinducer-3 (AI-3)/epinephrine (Epi)/norepinephrine (NE), and transduced by two-component systems QseBC has recently been described. As a fish pathogen and opportunistic pathogen for human beings, Edwardsiella tarda develops surface structures such as flagellar and fimbriae to cause different hemagglutination phenotypes and serotypes and initiate pathogen-host recognition and invasion process. E. tarda survives within macrophages in fish using type III secretion system (TTSS). Here, the genes of E. tarda two-component system, qseB and qseC, were found to be co-transcribed. Phylogenetic analysis indicated that evolution of QseC strongly correlated to different host niches. Compared with the wild type and their complemented strains, ΔqseB and ΔqseC mutants exhibited significant impaired flagellar motilities. Mammalian Epi was able to stimuli the flagellar motility in E. tarda via QseBC. Hemagglutination caused by fimbriae was induced in ΔqseB but repressed in ΔqseC. Disruption of qseB or qseC down-regulated the intracellular expressions of TTSS elements EseB and EsaC, and impaired their intracellular survival capabilities as well as in vivo competitive abilities. Furthermore, in vitro tests indicated that expression of EseB was induced by Epi via QseBC. Our results revealed that the QseBC system modified the virulence-related surface structures (flagellum, fimbriae and secretion system) and that hormone might stimulate the virulence of the pathogen in fish.     

QseC-mediated dephosphorylation of QseB is required for expression of genes associated with virulence in uropathogenic Escherichia coli

Bacteria sense environmental cues and regulate gene expression accordingly so as to persist in diverse niches. QseC is a membrane sensor kinase shown in enterohemorrhagic Escherichia coli to respond to host and bacterial signals by phosphorylating the QseB response regulator at residue D51, resulting in QseB activation and presumably upregulation of virulence genes. We studied QseBC in uropathogenic E. coli (UPEC). UPEC establish infection by colonizing and invading bladder cells. After invasion, UPEC can escape into the cytoplasm where they can form intracellular bacterial communities. Deletion of qseC significantly attenuated intracellular bacterial community formation and virulence, whereas paradoxically qseB deletion did not impact pathogenesis. We found that QseB upregulates its own expression in the qseC mutant, arguing that it is activated even in the absence of QseC. However, expression of QseB, but not a QseB_D51A mutant, in the absence of QseC resulted in downregulation of type 1 pili, curli and flagella. We observed similar phenotypes with enterohemorrhagic E. coli , showing that this is not a UPEC-specific phenomenon. Target gene expression is restored when QseC is present. We discovered that QseC has phosphatase activity required for QseB dephosphorylation. Thus, the QseC phosphatase capacity is critical for modulating QseB activity and subsequent gene expression.     

The impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and Vibrio harveyi on their virulence towards gnotobiotically cultured Artemia franciscana

Disruption of quorum sensing, bacterial cell-to-cell communication by means of small signal molecules, has been suggested as a new anti-infective strategy for aquaculture. However, data about the impact of quorum sensing on the virulence of aquatic pathogens are scarce. In this study, a model system using gnotobiotically cultured Artemia franciscana was developed in order to determine the impact of mutations in the quorum sensing systems of Aeromonas hydrophila, Vibrio anguillarum and V. harveyi on their virulence. Mutations in the autoinducer 2 (AI-2) synthase gene luxS, the AI-2 receptor gene luxP or the response regulator gene luxO of the dual channel quorum sensing system of V. harveyi abolished virulence of the strain towards Artemia. Moreover, the addition of an exogenous source of AI-2 could restore the virulence of an AI-2 non-producing mutant. In contrast, none of the mutations in either the acylated homoserine lactone (AHL)-mediated component of the V. harveyi system or the quorum sensing systems of Ae. hydrophila and V. anguillarum had an impact on virulence of these bacteria towards Artemia. Our results indicate that disruption of quorum sensing could be a good alternative strategy to combat infections caused by V. harveyi.     

luxS-dependent gene regulation in Escherichia coli K-12 revealed by genomic expression profiling

The bacterial quorum-sensing autoinducer 2 (AI-2) has received intense interest because the gene for its synthase, luxS, is common among a large number of bacterial species. We have identified luxS-controlled genes in Escherichia coli under two different growth conditions using DNA microarrays. Twenty-three genes were affected by luxS deletion in the presence of glucose, and 63 genes were influenced by luxS deletion in the absence of glucose. Minimal overlap among these gene sets suggests the role of luxS is condition dependent. Under the latter condition, the metE gene, the lsrACDBFG operon, and the flanking genes of the lsr operon (lsrR, lsrK, tam, and yneE) were among the most significantly induced genes by luxS. The E. coli lsr operon includes an additional gene, tam, encoding an S-adenosyl-l-methionine-dependent methyltransferase. Also, lsrR and lsrK belong to the same operon, lsrRK, which is positively regulated by the cyclic AMP receptor protein and negatively regulated by LsrR. lsrK is additionally transcribed by a promoter between lsrR and lsrK. Deletion of luxS was also shown to affect genes involved in methionine biosynthesis, methyl transfer reactions, iron uptake, and utilization of carbon. It was surprising, however, that so few genes were affected by luxS deletion in this E. coli K-12 strain under these conditions. Most of the highly induced genes are related to AI-2 production and transport. These data are consistent with the function of LuxS as an important metabolic enzyme but appear not to support the role of AI-2 as a true signal molecule for E. coli W3110 under the investigated conditions.     

A central metabolic circuit controlled by QseC in pathogenic Escherichia coli

The QseC sensor kinase regulates virulence in multiple Gram-negative pathogens, by controlling the activity of the QseB response regulator. We have previously shown that qseC deletion interferes with dephosphorylation of QseB thus unleashing what appears to be an uncontrolled positive feedback loop stimulating increased QseB levels. Deletion of QseC downregulates virulence gene expression and attenuates enterohaemorrhagic and uropathogenic Escherichia coli (EHEC and UPEC), Salmonella typhimurium, and Francisella tularensis. Given that these pathogens employ different infection strategies and virulence factors, we used genome-wide approaches to better understand the role of the QseBC interplay in pathogenesis. We found that deletion of qseC results in misregulation of nucleotide, amino acid, and carbon metabolism. Comparable metabolic changes are seen in EHEC ΔqseC, suggesting that deletion of qseC confers similar pleiotropic effects in these two different pathogens. Disruption of representative metabolic enzymes phenocopied UPEC ΔqseC in vivo and resulted in virulence factor downregulation. We thus propose that in the absence of QseC, the constitutively active QseB leads to pleiotropic effects, impairing bacterial metabolism, and thereby attenuating virulence. These findings provide a basis for the development of antimicrobials targeting the phosphatase activity of QseC, as a means to attenuate a wide range of QseC-bearing pathogens.     

Quorum signaling via AI-2 communicates the "Metabolic Burden" associated with heterologous protein production in Escherichia coli.

Recent reports have shown that bacterial cell-cell communication or quorum sensing is quite prevalent in pathogenic Escherichia coli, especially at high cell density; however, the role of quorum sensing in nonpathogenic E. coli is less clear and, in particular, there is no information regarding the role of quorum sensing in overexpression of plasmid-encoded genes. In this work, it was found that the activity of a quorum signaling molecule, autoinducer-2 (AI-2), decreased significantly following induction of several plasmid-encoded genes in both low and high-cell-density cultures of E. coli. Furthermore, we show that AI-2 signaling level was linearly related to the accumulation level of each protein product and that, in general, the highest rates of recombinant protein accumulation resulted in the greatest attenuation of AI-2 signaling. Importantly, our findings demonstrate for the first time that recombinant E. coli communicate the stress or burden of overexpressing heterologous genes through the quorum-based AI-2 signaling pathway.