Interspecific Quorum Sensing Mediates the Resuscitation of Viable but Nonculturable Vibrios

ABSTRACTEntry and exit from dormancy are essential survival mechanisms utilized by microorganisms to cope with harsh environments. Many bacteria, including the opportunistic human pathogenVibrio vulnificus, enter a form of dormancy known as the viable but nonculturable (VBNC) state. VBNC cells can resuscitate when suitable conditions arise, yet the molecular mechanisms facilitating resuscitation in most bacteria are not well understood. We discovered that bacterial cell-free supernatants (CFS) can awaken preexisting dormant vibrio populations within oysters and seawater, while CFS from a quorum sensing mutant was unable to produce the same resuscitative effect. Furthermore, the quorum sensing autoinducer AI-2 could induce resuscitation of VBNCV. vulnificus in vitro, and VBNC cells of a mutant unable to produce AI-2 were unable to resuscitate unless the cultures were supplemented with exogenous AI-2. The quorum sensing inhibitor cinnamaldehyde delayed the resuscitation of wild-type VBNC cells, confirming the importance of quorum sensing in resuscitation. By monitoring AI-2 production by VBNC cultures over time, we found quorum sensing signaling to be critical for the natural resuscitation process. This study provides new insights into the molecular mechanisms stimulating VBNC cell exit from dormancy, which has significant implications for microbial ecology and public health.

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LuxR Homologue SmcR Is Essential for Vibrio vulnificus Pathogenesis and Biofilm Detachment, and Its Expression is Induced by Host Cells

Infection and Immunity ◽

10.1128/iai.00561-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3721-3730 ◽

Cited By ~ 37

Author(s):

Seung Min Kim ◽

Jin Hwan Park ◽

Hyun Sung Lee ◽

Won Bin Kim ◽

Jung Min Ryu ◽

...

Keyword(s):

Quorum Sensing ◽

Vibrio Vulnificus ◽

Cell Communication ◽

Cellular Level ◽

Host Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Content Type ◽

Biofilm Detachment

ABSTRACTQuorum sensing is a cell-to-cell communication system known to control many bacterial processes. In the present study, the functions of quorum sensing in the pathogenesis ofVibrio vulnificus, a food-borne pathogen, were assessed by evaluating the virulence of a mutant deficient in SmcR, a quorum-sensing regulator and homologue of LuxR. When biofilms were used as an inoculum, thesmcRmutant was impaired in virulence and colonization capacity in the infection of mice. The lack of SmcR also resulted in decreased histopathological damage in mouse jejunum tissue. These results indicated that SmcR is essential forV. vulnificuspathogenesis. Moreover, thesmcRmutant exhibited significantly reduced biofilm detachment. Upon exposure to INT-407 host cells, the wild type, but not thesmcRmutant, revealed accelerated biofilm detachment. The INT-407 cells increasedsmcRexpression by activating the expression of LuxS, an autoinducer-2 synthase, indicating that host cells manipulate the cellular level of SmcR through the quorum-sensing signaling ofV. vulnificus. A whole-genome microarray analysis revealed that the genes primarily involved in biofilm detachment and formation are up- and downregulated by SmcR, respectively. Among the SmcR-regulated genes,vvpEencoding an elastolytic protease was the most upregulated, and the purified VvpE appeared to dissolve established biofilms directly in a concentration-dependent mannerin vitro. These results suggest that the host cell-induced SmcR enhances the detachment ofV. vulnificusbiofilms entering the host intestine and thereby may promote the dispersal of the pathogen to new colonization loci, which is crucial for pathogenesis.

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A Novel Infection Protocol in Zebrafish Embryo to Assess Pseudomonas aeruginosa Virulence and Validate Efficacy of a Quorum Sensing Inhibitor In Vivo

Pathogens ◽

10.3390/pathogens10040401 ◽

2021 ◽

Vol 10 (4) ◽

pp. 401

Author(s):

Pauline Nogaret ◽

Fatima El El Garah ◽

Anne-Béatrice Blanc-Potard

Keyword(s):

Pseudomonas Aeruginosa ◽

Animal Model ◽

Quorum Sensing ◽

Drug Testing ◽

Drug Efficacy ◽

Embryo Viability ◽

Immersion Method ◽

Opportunistic Human Pathogen

The opportunistic human pathogen Pseudomonas aeruginosa is responsible for a variety of acute infections and is a major cause of mortality in chronically infected cystic fibrosis patients. Due to increased resistance to antibiotics, new therapeutic strategies against P. aeruginosa are urgently needed. In this context, we aimed to develop a simple vertebrate animal model to rapidly assess in vivo drug efficacy against P. aeruginosa. Zebrafish are increasingly considered for modeling human infections caused by bacterial pathogens, which are commonly microinjected in embryos. In the present study, we established a novel protocol for zebrafish infection by P. aeruginosa based on bath immersion in 96-well plates of tail-injured embryos. The immersion method, followed by a 48-hour survey of embryo viability, was first validated to assess the virulence of P. aeruginosa wild-type PAO1 and a known attenuated mutant. We then validated its relevance for antipseudomonal drug testing by first using a clinically used antibiotic, ciprofloxacin. Secondly, we used a novel quorum sensing (QS) inhibitory molecule, N-(2-pyrimidyl)butanamide (C11), the activity of which had been validated in vitro but not previously tested in any animal model. A significant protective effect of C11 was observed on infected embryos, supporting the ability of C11 to attenuate in vivo P. aeruginosa pathogenicity. In conclusion, we present here a new and reliable method to compare the virulence of P. aeruginosa strains in vivo and to rapidly assess the efficacy of clinically relevant drugs against P. aeruginosa, including new antivirulence compounds.

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In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00310-19 ◽

2019 ◽

Vol 63 (10) ◽

Author(s):

Seong Eun Kim ◽

Hee Kyung Kim ◽

Su-Mi Choi ◽

Yohan Yu ◽

Uh Jin Kim ◽

...

Keyword(s):

Survival Rate ◽

Mortality Rate ◽

Combination Therapy ◽

Combination Treatment ◽

Vibrio Vulnificus ◽

Antibiotic Regimen ◽

Content Type ◽

Time Kill

ABSTRACT The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

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DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

Infection and Immunity ◽

10.1128/iai.00222-16 ◽

2016 ◽

Vol 84 (6) ◽

pp. 1879-1886 ◽

Cited By ~ 5

Author(s):

Lena J. Heung ◽

Tobias M. Hohl

Keyword(s):

Immune Response ◽

Nk Cells ◽

Cryptococcus Neoformans ◽

Molecular Mechanisms ◽

Content Type ◽

Effector Cells ◽

Opportunistic Fungal Pathogen ◽

Efficient Uptake ◽

Bacteria And Fungi

Cryptococcus neoformansis an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response toC. neoformans. Infectious outcomes in DAP12−/−mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12−/−mice. In contrast to WT NK cells, DAP12−/−NK cells are able to repressC. neoformansgrowthin vitro. Additionally, DAP12−/−macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing ofC. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response toC. neoformansby promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.

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Candida albicans Impacts Staphylococcus aureus Alpha-Toxin Production via Extracellular Alkalinization

mSphere ◽

10.1128/msphere.00780-19 ◽

2019 ◽

Vol 4 (6) ◽

Cited By ~ 3

Author(s):

Olivia A. Todd ◽

Mairi C. Noverr ◽

Brian M. Peters

Keyword(s):

Staphylococcus Aureus ◽

Candida Albicans ◽

Quorum Sensing ◽

Bacterial Pathogen ◽

Toxin Production ◽

Extracellular Ph ◽

Morbidity And Mortality ◽

Alpha Toxin ◽

Content Type

ABSTRACT Candida albicans and Staphylococcus aureus are common causes of nosocomial infections with severe morbidity and mortality. Murine polymicrobial intra-abdominal infection (IAI) with C. albicans and S. aureus results in acute mortality dependent on the secreted cytolytic effector alpha-toxin. Here, we confirmed that alpha-toxin is elevated during polymicrobial growth compared to monomicrobial growth in vitro. Therefore, this study sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal alpha-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced alpha-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and alpha-toxin production. Modulation of the pH could predictably attenuate or activate agr activity during coculture. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and alpha-toxin production during coculture. Overall, we demonstrate that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. IMPORTANCE Candida albicans and Staphylococcus aureus are commonly coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Thus, they represent a significant cause of nosocomial morbidity and mortality. Yet how these organisms behave in the context of polymicrobial growth remains poorly understood. In this work, we set out to determine the mechanism by which activation of the staphylococcal agr quorum sensing system and production of its major virulence effector alpha-toxin is enhanced during coculture with C. albicans. Surprisingly, we likely ruled out that a secreted candidal factor drives this process. Instead, we demonstrated that alkalinization of the extracellular milieu by C. albicans and other Candida species correlated with elevated agr activity. Thus, we propose a mechanism where modulation of the extracellular pH by fungal opportunists can indirectly alter virulence of a bacterial pathogen. Uncovering molecular events that drive interkingdom pathogenicity mechanisms may enhance surveillance and treatment for devastating polymicrobial infections.

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Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin

Applied and Environmental Microbiology ◽

10.1128/aem.00753-15 ◽

2015 ◽

Vol 81 (18) ◽

pp. 6158-6165 ◽

Cited By ~ 13

Author(s):

Tiffany C. Williams ◽

Mesrop Ayrapetyan ◽

James D. Oliver

Keyword(s):

Quorum Sensing ◽

Vibrio Vulnificus ◽

The United States ◽

Type Iv Pili ◽

Negative Regulator ◽

Physical Factors ◽

Wound Infections ◽

Environmental Persistence ◽

Content Type ◽

Type Iv

ABSTRACTThe human pathogenVibrio vulnificusis the leading cause of seafood-related deaths in the United States. Strains are genotyped on the basis of alleles that correlate with isolation source, with clinical (C)-genotype strains being more often implicated in disease and environmental (E)-genotype strains being more frequently isolated from oysters and estuarine waters. Previously, we have shown that the ecologically distinct C- and E-genotype strains ofV. vulnificusdisplay different degrees of chitin attachment, with C-genotype strains exhibiting reduced attachment relative to their E-genotype strain counterparts. We identified type IV pili to be part of the molecular basis for this observed genotypic variance, as E-genotype strains exhibit higher levels of expression of these genes than C-genotype strains. Here, we used a C-genotype quorum-sensing (QS) mutant to demonstrate that quorum sensing is a negative regulator of type IV pilus expression, which results in decreased chitin attachment. Furthermore, calcium depletion reduced E-genotype strain attachment to chitin, which suggests that calcium is necessary for proper functioning of the type IV pili in E-genotype strains. We also found that starvation or dormancy can alter the efficiency of chitin attachment, which has significant implications for the environmental persistence ofV. vulnificus. With the increasing incidence of wound infections caused byV. vulnificus, we investigated a subset of E-genotype strains isolated from human wound infections and discovered that they attached to chitin in a manner more similar to that of C-genotype strains. This study enhances our understanding of the molecular and physical factors that mediate chitin attachment inV. vulnificus, providing insight into the mechanisms that facilitate the persistence of this pathogen in its native environment.

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Complex Control of a Genomic Island Governing Biofilm and Rugose Colony Development in Vibrio vulnificus

Journal of Bacteriology ◽

10.1128/jb.00190-18 ◽

2018 ◽

Vol 200 (16) ◽

Cited By ~ 5

Author(s):

Daniel M. Chodur ◽

Dean A. Rowe-Magnus

Keyword(s):

Food Chain ◽

Horizontal Transfer ◽

Vibrio Vulnificus ◽

Regulatory Control ◽

Colony Development ◽

Dependent Manner ◽

Human Pathogen ◽

Content Type ◽

Regulatory Pathways ◽

Opportunistic Human Pathogen

ABSTRACT Vibrio vulnificus is a potent opportunistic human pathogen that contaminates the human food chain by asymptomatically colonizing seafood. The expression of the 9-gene brp exopolysaccharide locus mediates surface adherence and is controlled by the secondary signaling molecule c-di-GMP and the regulator BrpT. Here, we show that c-di-GMP and BrpT also regulate the expression of an adjacent 5-gene cluster that includes the cabABC operon, brpT, and another VpsT-like transcriptional regulator gene, brpS. The expression of the 14 genes spanning the region increased with elevated intracellular c-di-GMP levels in a BrpT-dependent manner, save for brpS, which was positively regulated by c-di-GMP and repressed by BrpT. BrpS repressed brpA expression and was required for rugose colony development. The mutation of its consensus WFSA c-di-GMP binding motif blocked these activities, suggesting that BrpS function is dependent on binding c-di-GMP. BrpT specifically bound the cabA, brpT, and brpS promoters, and binding sites homologous to the Vibrio cholerae VpsT binding site were identified upstream of brpA and brpT. Transcription was initiated distal to brpA, and a conserved RfaH-recruiting ops element and a potential Rho utilization (rut) terminator site were identified within the 100-bp leader region, suggesting the integration of early termination and operon polarity suppression into the regulation of brp transcription. The GC content and codon usage of the 16-kb brp region was 5.5% lower relative to that of the flanking DNA, suggesting its recent assimilation via horizontal transfer. Thus, architecturally, the brp region can be considered an acquired biofilm and rugosity island that is subject to complex regulation. IMPORTANCE Biofilm and rugose colony formation are developmental programs that underpin the evolution of Vibrio vulnificus as a potent opportunistic human pathogen and successful environmental organism. A better understanding of the regulatory pathways governing theses phenotypes promotes the development and implementation of strategies to mitigate food chain contamination by this pathogen. c-di-GMP signaling is central to both pathways. We show that the molecule orchestrates the expression of 14 genes clustered in a 16-kb segment of the genome that governs biofilm and rugose colony development. This region exhibits the hallmarks of horizontal transfer, suggesting complex regulatory control of a recently assimilated genetic island governing the colonization response of V. vulnificus.

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Hierarchical Transcriptional Control of the LuxR Quorum-Sensing Regulon of Vibrio harveyi

Journal of Bacteriology ◽

10.1128/jb.00047-20 ◽

2020 ◽

Vol 202 (14) ◽

Author(s):

Ryan R. Chaparian ◽

Alyssa S. Ball ◽

Julia C. van Kessel

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Quorum Sensing ◽

Cell Density ◽

Regulatory Networks ◽

Vibrio Harveyi ◽

Sugar Transport ◽

Content Type ◽

Second Tier

ABSTRACT In vibrios, quorum sensing controls hundreds of genes that are required for cell density-specific behaviors including bioluminescence, biofilm formation, competence, secretion, and swarming motility. The central transcription factor in the quorum-sensing pathway is LuxR/HapR, which directly regulates ∼100 genes in the >400-gene regulon of Vibrio harveyi. Among these directly controlled genes are 15 transcription factors, which we predicted would comprise the second tier in the hierarchy of the LuxR regulon. We confirmed that LuxR binds to the promoters of these genes in vitro and quantified the extent of LuxR activation or repression of transcript levels. Transcriptome sequencing (RNA-seq) indicates that most of these transcriptional regulators control only a few genes, with the exception of MetJ, which is a global regulator. The genes regulated by these transcription factors are predicted to be involved in methionine and thiamine biosynthesis, membrane stability, RNA processing, c-di-GMP degradation, sugar transport, and other cellular processes. These data support a hierarchical model in which LuxR directly regulates 15 transcription factors that drive the second level of the gene expression cascade to influence cell density-dependent metabolic states and behaviors in V. harveyi. IMPORTANCE Quorum sensing is important for survival of bacteria in nature and influences the actions of bacterial groups. In the relatively few studied examples of quorum-sensing-controlled genes, these genes are associated with competition or cooperation in complex microbial communities and/or virulence in a host. However, quorum sensing in vibrios controls the expression of hundreds of genes, and their functions are mostly unknown or uncharacterized. In this study, we identify the regulators of the second tier of gene expression in the quorum-sensing system of the aquaculture pathogen Vibrio harveyi. Our identification of regulatory networks and metabolic pathways controlled by quorum sensing can be extended and compared to other Vibrio species to understand the physiology, ecology, and pathogenesis of these organisms.

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The 5′ NAD Cap of RNAIII Modulates Toxin Production in Staphylococcus aureus Isolates

Journal of Bacteriology ◽

10.1128/jb.00591-19 ◽

2019 ◽

Vol 202 (6) ◽

Cited By ~ 3

Author(s):

Hector Gabriel Morales-Filloy ◽

Yaqing Zhang ◽

Gabriele Nübel ◽

Shilpa Elizabeth George ◽

Natalya Korn ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Secondary Structure ◽

Quorum Sensing ◽

Opportunistic Pathogen ◽

Toxin Production ◽

Dual Function ◽

Content Type ◽

The Stability

ABSTRACT Nicotinamide adenosine dinucleotide (NAD) has been found to be covalently attached to the 5′ ends of specific RNAs in many different organisms, but the physiological consequences of this modification are largely unknown. Here, we report the occurrence of several NAD-RNAs in the opportunistic pathogen Staphylococcus aureus. Most prominently, RNAIII, a central quorum-sensing regulator of this bacterium’s physiology, was found to be 5′ NAD capped in a range from 10 to 35%. NAD incorporation efficiency into RNAIII was found to depend in vivo on the −1 position of the P3 promoter. An increase in RNAIII’s NAD content led to a decreased expression of alpha- and delta-toxins, resulting in reduced cytotoxicity of the modified strains. These effects seem to be caused neither by changes in RNAIII’s secondary structure nor by a different translatability upon NAD attachment, as indicated by unaltered patterns in in vitro chemical probing and toeprinting experiments. Even though we did not observe any effect of this modification on RNAIII’s secondary structure or translatability in vitro, additional unidentified factors might account for the modulation of exotoxins in vivo. Ultimately, the study constitutes a step forward in the discovery of new roles of the NAD molecule in bacteria. IMPORTANCE Numerous organisms, including bacteria, are endowed with a 5′ NAD cap in specific RNAs. While the presence of the 5′ NAD cap modulates the stability of the modified RNA species, a significant biological function and phenotype have not been assigned so far. Here, we show the presence of a 5′ NAD cap in RNAIII from S. aureus, a dual-function regulatory RNA involved in quorum-sensing processes and regulation of virulence factor expression. We also demonstrate that altering the natural NAD modification ratio of RNAIII leads to a decrease in exotoxin production, thereby modulating the bacterium’s virulence. Our work unveils a new layer of regulation of RNAIII and the agr system that might be linked to the redox state of the NAD molecule in the cell.

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A rhlI 5′ UTR-Derived sRNA Regulates RhlR-Dependent Quorum Sensing in Pseudomonas aeruginosa

mBio ◽

10.1128/mbio.02253-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 5

Author(s):

Maureen K. Thomason ◽

Maya Voichek ◽

Daniel Dar ◽

Victoria Addis ◽

David Fitzgerald ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Homoserine Lactone ◽

Regulatory Elements ◽

Content Type ◽

Reading Frame ◽

Bacterial Physiology ◽

Small Regulatory Rna ◽

Key Enzyme ◽

Opportunistic Human Pathogen

ABSTRACT N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5′ untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS. IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.

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