Vibrio cholerae LeuO Links the ToxR Regulon to Expression of Lipid A Remodeling Genes

Vibrio choleraeis an intestinal pathogen that causes the diarrheal disease cholera. Colonization of the intestine depends upon the expression of genes that allowV. choleraeto overcome host barriers, including low pH, bile acids, and the innate immune system. ToxR is a major contributor to this process. ToxR is a membrane-spanning transcription factor that coordinates gene expression in response to environmental cues. In previous work we showed that ToxR upregulatedleuOexpression in response to bile salts. LeuO is a LysR family transcription factor that contributes to acid tolerance, bile resistance, and biofilm formation inV. cholerae. Here, we investigated the function of ToxR and LeuO in cationic antimicrobial peptide (CAMP) resistance. We report that ToxR and LeuO contribute to CAMP resistance by regulatingcarRStranscription. CarRS is a two-component regulatory system that positively regulatesalmEFGexpression. AlmEFG confers CAMP resistance by glycinylation of lipid A. We found that the expression ofcarRSandalmEFGand the polymyxin B MIC increased in mutants lackingtoxRSorleuO. Conversely,leuOoverexpression decreased the polymyxin B MIC. Furthermore, we found that LeuO directly bound to thecarRSpromoter and that ToxR-dependent activation ofleuOtranscription regulatedcarRStranscription in response to bile salts. Our results suggest that LeuO functions downstream of ToxR to modulatecarRSexpression in response to environmental cues. This study extends the functional role of ToxR and LeuO in environmental adaptation to include cell surface remodeling and CAMP resistance.

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Polymyxin B Resistance and Biofilm Formation in Vibrio cholerae Are Controlled by the Response Regulator CarR

Infection and Immunity ◽

10.1128/iai.02700-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1199-1209 ◽

Cited By ~ 38

Author(s):

Kivanc Bilecen ◽

Jiunn C. N. Fong ◽

Andrew Cheng ◽

Christopher J. Jones ◽

David Zamorano-Sánchez ◽

...

Keyword(s):

Vibrio Cholerae ◽

Biofilm Formation ◽

Lipid A ◽

Response Regulator ◽

Regulatory Region ◽

Regulatory System ◽

Polymyxin B ◽

Content Type ◽

Two Component ◽

Antimicrobial Peptide Resistance

Two-component systems play important roles in the physiology of many bacterial pathogens.Vibrio cholerae's CarRS two-component regulatory system negatively regulates expression ofvps(Vibriopolysaccharide) genes and biofilm formation. In this study, we report that CarR confers polymyxin B resistance by positively regulating expression of thealmEFGgenes, whose products are required for glycine and diglycine modification of lipid A. We determined that CarR directly binds to the regulatory region of thealmEFGoperon. Similarly to acarRmutant, strains lackingalmE,almF, andalmGexhibited enhanced polymyxin B sensitivity. We also observed that strains lackingalmEor thealmEFGoperon have enhanced biofilm formation. Our results reveal that CarR regulates biofilm formation and antimicrobial peptide resistance inV. cholerae.

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Calcium Enhances Bile Salt-Dependent Virulence Activation in Vibrio cholerae

Infection and Immunity ◽

10.1128/iai.00707-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 7

Author(s):

Amanda J. Hay ◽

Menghua Yang ◽

Xiaoyun Xia ◽

Zhi Liu ◽

Justin Hammons ◽

...

Keyword(s):

Bile Salt ◽

Vibrio Cholerae ◽

Bile Salts ◽

Diarrheal Disease ◽

Content Type ◽

Inner Membrane Protein ◽

Virulence Regulator ◽

Host Signals

ABSTRACT Vibrio cholerae is the causative bacteria of the diarrheal disease cholera, but it also persists in aquatic environments, where it displays an expression profile that is distinct from that during infection. Upon entry into the host, a tightly regulated circuit coordinates the induction of two major virulence factors: cholera toxin and a toxin-coregulated pilus (TCP). It has been shown that a set of bile salts, including taurocholate, serve as host signals to activate V. cholerae virulence through inducing the activity of the transmembrane virulence regulator TcpP. In this study, we investigated the role of calcium, an abundant mental ion in the gut, in the regulation of virulence. We show that whereas Ca2+ alone does not affect virulence, Ca2+ enhances bile salt-dependent virulence activation for V. cholerae. The induction of TCP by murine intestinal contents is counteracted when Ca2+ is depleted by the high-affinity calcium chelator EGTA, suggesting that the calcium present in the gut is a relevant signal for V. cholerae virulence induction in vivo. We further show that Ca2+ enhances virulence by promoting bile salt-induced TcpP-TcpP interaction. Moreover, fluorescence recovery after photobleaching (FRAP) analysis demonstrated that exposure to bile salts and Ca2+ together decreases the recovery rate for fluorescently labeled TcpP, but not for another inner membrane protein (TatA). Together, these data support a model in which physiological levels of Ca2+ may result in altered bile salt-induced TcpP protein movement and activity, ultimately leading to an increased expression of virulence.

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A Point Mutation in carR Is Involved in the Emergence of Polymyxin B-Sensitive Vibrio cholerae O1 El Tor Biotype by Influencing Gene Transcription

Infection and Immunity ◽

10.1128/iai.00080-20 ◽

2020 ◽

Vol 88 (5) ◽

Cited By ~ 1

Author(s):

Prosenjit Samanta ◽

Rahul Shubhra Mandal ◽

Rudra Narayan Saha ◽

Sreeja Shaw ◽

Priyanka Ghosh ◽

...

Keyword(s):

Vibrio Cholerae ◽

Lipid A ◽

Polymyxin B ◽

Sensitive Strain ◽

Two Component System ◽

Content Type ◽

Vibrio Cholerae O1 ◽

Binding Cleft ◽

Culturing Conditions ◽

El Tor

ABSTRACT Antimicrobial peptides play an important role in host defense against Vibrio cholerae. Generally, the V. cholerae O1 classical biotype is polymyxin B (PB) sensitive and El Tor is relatively resistant. Detection of classical biotype traits like the production of classical cholera toxin and PB sensitivity in El Tor strains has been reported in recent years, including in the devastating Yemen cholera outbreak during 2016-2018. To investigate the factor(s) responsible for the shift in the trend of sensitivity to PB, we studied the two-component system encoded by carRS, regulating the lipid A modification of El Tor vibrios, and found that only carR contains a single nucleotide polymorphism (SNP) in recently emerged PB-sensitive strains. We designated the two alleles present in PB-resistant and -sensitive strains carRr and carRs alleles, respectively, and replaced the carRs allele of a sensitive strain with the carRr allele, using an allelic-exchange approach. The sensitive strain then became resistant. The PB-resistant strain N16961 was made susceptible to PB in a similar fashion. Our in silico CarR protein models suggested that the D89N substitution in the more stable CarRs protein brings the two structural domains of CarR closer, constricting the DNA binding cleft. This probably reduces the expression of the carR-regulated almEFG operon, inducing PB susceptibility. Expression of almEFG in PB-sensitive strains was found to be downregulated under natural culturing conditions. In addition, the expression of carR and almEG decreased in all strains with increased concentrations of extracellular Ca2+ but increased with a rise in pH. The downregulation of almEFG in CarRs strains confirmed that the G265A mutation is responsible for the emergence of PB-sensitive El Tor strains.

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The MisR Response Regulator Is Necessary for Intrinsic Cationic Antimicrobial Peptide and Aminoglycoside Resistance in Neisseria gonorrhoeae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00823-16 ◽

2016 ◽

Vol 60 (8) ◽

pp. 4690-4700 ◽

Cited By ~ 13

Author(s):

Justin L. Kandler ◽

Concerta L. Holley ◽

Jennifer L. Reimche ◽

Vijaya Dhulipala ◽

Jacqueline T. Balthazar ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Lipid A ◽

Efflux Pump ◽

Response Regulator ◽

Membrane Integrity ◽

Regulatory System ◽

Phagocytic Cells ◽

Aminoglycoside Resistance ◽

Cationic Antimicrobial Peptide ◽

Content Type

ABSTRACTDuring infection, the sexually transmitted pathogenNeisseria gonorrhoeae(the gonococcus) encounters numerous host-derived antimicrobials, including cationic antimicrobial peptides (CAMPs) produced by epithelial and phagocytic cells. CAMPs have both direct and indirect killing mechanisms and help link the innate and adaptive immune responses during infection. Gonococcal CAMP resistance is likely important for avoidance of host nonoxidative killing systems expressed by polymorphonuclear granulocytes (e.g., neutrophils) and intracellular survival. Previously studied gonococcal CAMP resistance mechanisms include modification of lipid A with phosphoethanolamine by LptA and export of CAMPs by the MtrCDE efflux pump. In the related pathogenNeisseria meningitidis, a two-component regulatory system (2CRS) termed MisR-MisS has been shown to contribute to the capacity of the meningococcus to resist CAMP killing. We report that the gonococcal MisR response regulator but not the MisS sensor kinase is involved in constitutive and inducible CAMP resistance and is also required for intrinsic low-level resistance to aminoglycosides. The 4- to 8-fold increased susceptibility ofmisR-deficient gonococci to CAMPs and aminoglycosides was independent of phosphoethanolamine decoration of lipid A and the levels of the MtrCDE efflux pump and seemed to correlate with a general increase in membrane permeability. Transcriptional profiling and biochemical studies confirmed that expression oflptAandmtrCDEwas not impacted by the loss of MisR. However, several genes encoding proteins involved in membrane integrity and redox control gave evidence of being MisR regulated. We propose that MisR modulates the levels of gonococcal susceptibility to antimicrobials by influencing the expression of genes involved in determining membrane integrity.

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New Aspects of RpoE in Uropathogenic Proteus mirabilis

Infection and Immunity ◽

10.1128/iai.02232-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 966-977 ◽

Cited By ~ 10

Author(s):

Ming-Che Liu ◽

Kuan-Ting Kuo ◽

Hsiung-Fei Chien ◽

Yi-Lin Tsai ◽

Shwu-Jen Liaw

Keyword(s):

Urinary Tract ◽

Virulence Factors ◽

Stress Responses ◽

Proteus Mirabilis ◽

Immune Cell ◽

Polymyxin B ◽

Urothelial Cells ◽

Cationic Antimicrobial Peptide ◽

Content Type ◽

Tract Infections

Proteus mirabilisis a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms forP. mirabilisto establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulatingmrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted fromrpoEmutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness ofP. mirabilisin the host, including the avoidance of immune attacks. Accordingly,rpoEmutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and therpoEmutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression ofrpoEby the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness ofP. mirabilis, to build up a UTI.

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Polymyxin B Resistance in El Tor Vibrio cholerae Requires Lipid Acylation Catalyzed by MsbB

Journal of Bacteriology ◽

10.1128/jb.00023-10 ◽

2010 ◽

Vol 192 (8) ◽

pp. 2044-2052 ◽

Cited By ~ 31

Author(s):

Jyl S. Matson ◽

Hyun Ju Yoo ◽

Kristina Hakansson ◽

Victor J. DiRita

Keyword(s):

Antimicrobial Peptides ◽

Vibrio Cholerae ◽

Lipid A ◽

Polymyxin B ◽

Mass Spectrometry Analysis ◽

Peptide Antibiotic ◽

Wild Type ◽

Spectrometry Analysis ◽

Antimicrobial Peptide Resistance ◽

El Tor

ABSTRACTAntimicrobial peptides are critical for innate antibacterial defense. Both Gram-negative and Gram-positive microbes have mechanisms to alter their surfaces and resist killing by antimicrobial peptides. InVibrio cholerae, two natural epidemic biotypes, classical and El Tor, exhibit distinct phenotypes with respect to sensitivity to the peptide antibiotic polymyxin B: classical strains are sensitive and El Tor strains are relatively resistant. We carried out mutant screens of both biotypes, aiming to identify classicalV. choleraemutants resistant to polymyxin B and El TorV. choleraemutants sensitive to polymyxin B. Insertions in a gene annotatedmsbB(encoding a predicted lipid A secondary acyltransferase) answered both screens, implicating its activity in antimicrobial peptide resistance ofV. cholerae. Analysis of a defined mutation in the El Tor biotype demonstrated thatmsbBis required for resistance to all antimicrobial peptides tested. Mutation ofmsbBin a classical strain resulted in reduced resistance to several antimicrobial peptides but in no significant change in resistance to polymyxin B.msbBmutants of both biotypes showed decreased colonization of infant mice, with a more pronounced defect observed for the El Tor mutant. Mass spectrometry analysis showed that lipid A of themsbBmutant for both biotypes was underacylated compared to lipid A of the wild-type isolates, confirming that MsbB is a functional acyltransferase inV. cholerae.

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2-Hydroxylation ofAcinetobacter baumanniiLipid A Contributes to Virulence

Infection and Immunity ◽

10.1128/iai.00066-19 ◽

2019 ◽

Vol 87 (4) ◽

Cited By ~ 6

Author(s):

Toby L. Bartholomew ◽

Timothy J. Kidd ◽

Joana Sá Pessoa ◽

Raquel Conde Álvarez ◽

José A. Bengoechea

Keyword(s):

Protein Kinase ◽

Lipid A ◽

Galleria Mellonella ◽

Inflammatory Responses ◽

Polymyxin B ◽

Interleukin 10 ◽

Multidrug Resistant ◽

Mitogen Activated Protein Kinase ◽

Content Type ◽

Wide Range

ABSTRACTAcinetobacter baumanniicauses a wide range of nosocomial infections. This pathogen is considered a threat to human health due to the increasingly frequent isolation of multidrug-resistant strains. There is a major gap in knowledge on the infection biology ofA. baumannii, and only a few virulence factors have been characterized, including lipopolysaccharide. The lipid A expressed byA. baumanniiis hepta-acylated and contains 2-hydroxylaurate. The late acyltransferases controlling the acylation of lipid A have been already characterized. Here, we report the characterization ofA. baumanniiLpxO, which encodes the enzyme responsible for the 2-hydroxylation of lipid A. By genetic methods and mass spectrometry, we demonstrate that LpxO catalyzes the 2-hydroxylation of the laurate transferred byA. baumanniiLpxL. LpxO-dependent lipid A 2-hydroxylation protectsA. baumanniifrom polymyxin B, colistin, and human β-defensin 3. LpxO contributes to the survival ofA. baumanniiin human whole blood and is required for pathogen survival in the waxmothGalleria mellonella. LpxO also protectsAcinetobacterfromG. mellonellaantimicrobial peptides and limits their expression. Further demonstrating the importance of LpxO-dependent modification in immune evasion, 2-hydroxylation of lipid A limits the activation of the mitogen-activated protein kinase Jun N-terminal protein kinase to attenuate inflammatory responses. In addition, LpxO-controlled lipid A modification mediates the production of the anti-inflammatory cytokine interleukin-10 (IL-10) via the activation of the transcriptional factor CREB. IL-10 in turn limits the production of inflammatory cytokines followingA. baumanniiinfection. Altogether, our studies suggest that LpxO is a candidate for the development of anti-A. baumanniidrugs.

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Alterations of Metabolic and Lipid Profiles in Polymyxin-ResistantPseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02656-17 ◽

2018 ◽

Vol 62 (6) ◽

Cited By ~ 15

Author(s):

Mei-Ling Han ◽

Yan Zhu ◽

Darren J. Creek ◽

Yu-Wei Lin ◽

Dovile Anderson ◽

...

Keyword(s):

Lipid A ◽

Therapeutic Option ◽

Polymyxin B ◽

Multidrug Resistant ◽

Wild Type ◽

Metabolomics Data ◽

Content Type ◽

In The Wild ◽

High Level ◽

Acyl Coenzyme A

ABSTRACTMultidrug-resistantPseudomonas aeruginosapresents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance inP. aeruginosahas been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistantP. aeruginosastrains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-typeP. aeruginosastrain K ([PAK] polymyxin B MIC, 1 mg/liter) and its pairedpmrBmutant strains, PAKpmrB6and PAKpmrB12(polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) inspeE(encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6compared to that in PAKpmrB12. Our results indicate that spermidine may play an important role in high-level polymyxin resistance inP. aeruginosa. Interestingly, bothpmrBmutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12mutant exhibited much lower levels of phospholipids than the PAKpmrB6mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance inP. aeruginosaand highlights its impacts on bacterial metabolism.

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Neutrophils Are Essential for Containment of Vibrio cholerae to the Intestine during the Proinflammatory Phase of Infection

Infection and Immunity ◽

10.1128/iai.00356-12 ◽

2012 ◽

Vol 80 (8) ◽

pp. 2905-2913 ◽

Cited By ~ 32

Author(s):

Jessica Queen ◽

Karla J. Fullner Satchell

Keyword(s):

Immune Response ◽

Vibrio Cholerae ◽

Diarrheal Disease ◽

Innate Immune ◽

Infection Model ◽

Proinflammatory Response ◽

Necrosis Factor Alpha ◽

Live Attenuated Vaccine ◽

Content Type

ABSTRACTCholera is classically considered a noninflammatory diarrheal disease, in comparison to invasive enteric organisms, although there is a low-level proinflammatory response during early infection withVibrio choleraeand a strong proinflammatory reaction to live attenuated vaccine strains. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host defense to infection. Nontoxigenic El Tor O1V. choleraeinfection is characterized by the upregulation of interleukin-6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha in the intestine, indicating an acute innate immune response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to decreased survival of mice. The role of neutrophils in protection of the host is to limit the infection to the intestine and control bacterial spread to extraintestinal organs. In the absence of neutrophils, the infection spread to the spleen and led to increased systemic levels of IL-1β and tumor necrosis factor alpha, suggesting the decreased survival in neutropenic mice is due to systemic shock. Neutrophils were found not to contribute to either clearance of colonizing bacteria or to alter the local immune response. However, when genes for secreted accessory toxins were deleted, the colonizing bacteria were cleared from the intestine, and this clearance is dependent upon neutrophils. Thus, the requirement for accessory toxins in virulence is negated in neutropenic mice, which is consistent with a role of accessory toxins in the evasion of innate immune cells in the intestine. Overall, these data support that neutrophils impact disease progression and suggest that neutrophil effectiveness can be manipulated through the deletion of accessory toxins.

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ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

Biochemical Journal ◽

10.1042/bj20111639 ◽

2012 ◽

Vol 442 (1) ◽

pp. 85-93 ◽

Cited By ~ 33

Author(s):

Corinne Appia-Ayme ◽

Andrea Hall ◽

Elaine Patrick ◽

Shiny Rajadurai ◽

Thomas A. Clarke ◽

...

Keyword(s):

Antimicrobial Agents ◽

Regulatory System ◽

Enteric Bacteria ◽

Polymyxin B ◽

Regulatory Function ◽

Cationic Antimicrobial Peptide ◽

Envelope Stress ◽

Two Component ◽

Periplasmic Proteins ◽

Zinc Concentrations

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

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