Byoung Sik Kim

Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon

Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorum-sensing signals for many Proteobacteria, and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa, which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorum-sensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa. As a test of our hypothesis we used RNA-seq to identify quorum-controlled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensing–regulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorum-controlled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.

The Capability of Catabolic Utilization of N-Acetylneuraminic Acid, a Sialic Acid, Is Essential for Vibrio vulnificus Pathogenesis

ABSTRACT N-acetylneuraminic acid (Neu5Ac, sialic acid) could provide a good substrate for enteropathogenic bacteria in the intestine, when the bacteria invade and colonize in human gut. In order to analyze the role of Neu5Ac catabolism in Vibrio vulnificus pathogenesis, a mutant with disruption of the nanA gene encoding Neu5Ac lyase was constructed by allelic exchanges. The nanA mutant was not able to utilize Neu5Ac as a sole carbon source and revealed an altered colony morphotype with reduced opacity in the presence of Neu5Ac. Compared to the wild type, the nanA mutant exhibited a low level of cytotoxicity toward INT-407 epithelial cells in vitro and reduced virulence in a mouse model. The disruption of nanA also resulted in a substantial decrease in histopathological damage in jejunum and colon tissues from the mouse intestine. These results indicated that NanA plays an important role in V. vulnificus pathogenesis. In addition, the nanA mutant was significantly diminished in growth with and adherence to INT-407 epithelial cells in vitro, and was defective for intestinal colonization, reflecting the impaired ability of the mutant to grow and survive with, persist in, and adhere to the intestine in vivo. Consequently, the combined results suggest that NanA and the capability of catabolic utilization of Neu5Ac contribute to V. vulnificus virulence by ensuring growth, adhesion, and survival during infection.

Vibrio vulnificus rtxE Is Important for Virulence, and Its Expression Is Induced by Exposure to Host Cells

ABSTRACT Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity to INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. A transposon-tagging method was used to identify and clone an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from Vibrio cholerae. Functions of the rtxE gene in virulence were assessed by constructing an isogenic mutant whose rtxE gene was inactivated by allelic exchanges and by evaluating the differences between its virulence phenotype and that of the wild type in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity against epithelial cells in vitro. Also, the intraperitoneal 50% lethal dose of the rtxE mutant was 104 to 105 times higher than that of the parental wild type, indicating that RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter, PrtxBDE. The activity of V. vulnificus PrtxBDE is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria with the host cells.

Cooperative Regulation of the Vibrio vulnificus nan Gene Cluster by NanR Protein, cAMP Receptor Protein, and N-Acetylmannosamine 6-Phosphate

Background: Catabolic utilization of sialic acid is essential for the pathogenesis of enteropathogens. Results: NanR, CRP, and ManNAc-6P regulate the V. vulnificus nan cluster required for catabolism of Neu5Ac, a sialic acid. Conclusion: This cooperative regulation leads to precise tuning of the nan cluster expression. Significance: The results shed insight into the understanding of sialate metabolism central to host-microbe interactions. The nan cluster of Vibrio vulnificus, a food-borne pathogen, consists of two divergently transcribed operons, nanTPSLAR and nanEK nagA, required for transport and catabolism of N-acetylneuraminic acid (Neu5Ac). A mutation of nanR abolished the extensive lag phase observed for the bacteria growing on Neu5Ac and increased transcription of nanTP and nanE, suggesting that NanR is a transcriptional repressor of both nan operons. Intracellular accumulation of Neu5Ac was dependent on the carbon source, implying that the nan operons are also subject to catabolite repression. Hence, cAMP receptor protein (CRP) appeared to activate and repress transcription of nanTPSLAR and nanEK nagA, respectively. Direct bindings of NanR and CRP to the nanTP-nanE intergenic DNA were demonstrated by EMSA. Two adjacent NanR-binding sites centered at +44.5 and −10 and a CRP-binding site centered at −60.5 from the transcription start site of nanTP were identified by DNase I protection assays. Mutagenesis approaches, in vitro transcription, and isothermal titration calorimetry experiments demonstrated that N-acetylmannosamine 6-phosphate specifically binds to NanR and functions as the inducer of the nan operons. The combined results propose a model in which NanR, CRP, and N-acetylmannosamine 6-phosphate cooperate for precise adjustment of the expression level of the V. vulnificus nan cluster.

Crystal Structure of SmcR, a Quorum-sensing Master Regulator of Vibrio vulnificus, Provides Insight into Its Regulation of Transcription

Quorum sensing has been implicated as an important global regulatory system controlling the expression of numerous virulence factors in bacterial pathogens. SmcR, a homologue of Vibrio harveyi LuxR, has been proposed as a quorum-sensing master regulator of Vibrio vulnificus, an opportunistic human pathogen. Previous studies demonstrated that SmcR is essential for the survival and pathogenesis of V. vulnificus, indicating that inhibiting SmcR is an attractive approach to combat infections by the bacteria. Here, we determined the crystal structure of SmcR at 2.1 Å resolution. The protein structure reveals a typical TetR superfamily fold consisting of an N-terminal DNA binding domain and a C-terminal dimerization domain. In vivo and in vitro functional analysis of the dimerization domain suggested that dimerization of SmcR is vital for its biological regulatory function. The N-terminal DNA recognition and binding residues were assigned based on the protein structure and the results of in vivo and in vitro mutagenesis experiments. Furthermore, protein-DNA interaction experiments suggested that SmcR may have a sophisticated mechanism that enables the protein to recognize each of its many target operators with different affinities.

Identification and Characterization of a Novel Serine Protease, VvpS, That Contains Two Functional Domains and Is Essential for Autolysis of Vibrio vulnificus

Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus, a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic β-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus. VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus.

Structural insights into the regulation of sialic acid catabolism by the Vibrio vulnificus transcriptional repressor NanR

Significance Pathogenic bacteria that experience limited nutrient availability in the host gut have evolved sophisticated systems to catabolize N-acetylneuraminic acid (Neu5Ac; sialic acid). This study reports the structural analysis of NanR, a repressor of the N-acetylneuraminate (nan) genes responsible for Neu5Ac catabolism, complexed with its regulatory ligand, N-acetylmannosamine 6-phosphate (ManNAc-6P). The interaction between NanR and the nan promoter is alleviated by the ManNAc-6P–mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of nan genes. These events are required for survival and for Vibrio vulnificus pathogenesis. Pathogenic and commensal bacteria that experience limited nutrient availability in their host have evolved sophisticated systems to catabolize the mucin sugar N-acetylneuraminic acid, thereby facilitating their survival and colonization. The correct function of the associated catabolic machinery is particularly crucial for the pathogenesis of enteropathogenic bacteria during infection, although the molecular mechanisms involved with the regulation of the catabolic machinery are unknown. This study reports the complex structure of NanR, a repressor of the N-acetylneuraminate (nan) genes responsible for N-acetylneuraminic acid catabolism, and its regulatory ligand, N-acetylmannosamine 6-phosphate (ManNAc-6P), in the human pathogenic bacterium Vibrio vulnificus. Structural studies combined with electron microscopic, biochemical, and in vivo analysis demonstrated that NanR forms a dimer in which the two monomers create an arched tunnel-like DNA-binding space, which contains positively charged residues that interact with the nan promoter. The interaction between the NanR dimer and DNA is alleviated by the ManNAc-6P–mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of the nan genes. Survival studies in which mice were challenged with a ManNAc-6P–binding-defective mutant strain of V. vulnificus demonstrated that this relocation of NanR residues is critical for V. vulnificus pathogenesis. In summary, this study presents a model of the mechanism that regulates sialic acid catabolism via NanR in V. vulnificus.

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase–dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA. Molecular biological analyses revealed that plpA expression is controlled via the promoter, PplpA, and that HlyU and CRP directly bind to PplpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

QStatin, a Selective Inhibitor of Quorum Sensing in Vibrio Species

ABSTRACT Pathogenic Vibrio species cause diseases in diverse marine animals reared in aquaculture. Since their pathogenesis, persistence, and survival in marine environments are regulated by quorum sensing (QS), QS interference has attracted attention as a means to control these bacteria in aquatic settings. A few QS inhibitors of Vibrio species have been reported, but detailed molecular mechanisms are lacking. Here, we identified a novel, potent, and selective Vibrio QS inhibitor, named QStatin [1-(5-bromothiophene-2-sulfonyl)-1H-pyrazole], which affects Vibrio harveyi LuxR homologues, the well-conserved master transcriptional regulators for QS in Vibrio species. Crystallographic and biochemical analyses showed that QStatin binds tightly to a putative ligand-binding pocket in SmcR, the LuxR homologue in V. vulnificus, and changes the flexibility of the protein, thereby altering its transcription regulatory activity. Transcriptome analysis revealed that QStatin results in SmcR dysfunction, affecting the expression of SmcR regulon required for virulence, motility/chemotaxis, and biofilm dynamics. Notably, QStatin attenuated representative QS-regulated phenotypes in various Vibrio species, including virulence against the brine shrimp (Artemia franciscana). Together, these results provide molecular insights into the mechanism of action of an effective, sustainable QS inhibitor that is less susceptible to resistance than other antimicrobial agents and useful in controlling the virulence of Vibrio species in aquacultures. IMPORTANCE Yields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenic Vibrio infections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis of Vibrio species in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor of V. harveyi LuxR homologues, which are well-conserved master QS regulators in Vibrio species. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue in V. vulnificus, and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in various Vibrio species, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures. IMPORTANCE Yields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenic Vibrio infections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis of Vibrio species in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor of V. harveyi LuxR homologues, which are well-conserved master QS regulators in Vibrio species. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue in V. vulnificus, and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in various Vibrio species, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures.