WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

ABSTRACTThe general stress response (GSR) system of the intracellular pathogenBrucella abortuscontrols the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required forB. abortussurvival under nonoptimal growth conditionsin vitroand for maintenance of chronic infection in anin vivomouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined.bab1_1070is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditionsin vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However,B. abortusWrbA-relatedprotein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductasein vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion ofwrpA(ΔwrpA) does not compromise cell survival under acute oxidative stressin vitroor attenuate infection in cell-based or mouse models. However, a ΔwrpAstrain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulatesB. abortusinteraction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose thatB. abortusWrpA represents a functionally distinct member of the diverse flavodoxin family.IMPORTANCEBrucella abortusis an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system ofB. abortuscontrols the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We presentin vitroandin vivofunctional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activityin vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

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The general stress response of Staphylococcus aureus promotes tolerance of antibiotics and survival in whole human blood

Microbiology ◽

10.1099/mic.0.000983 ◽

2020 ◽

Vol 166 (11) ◽

pp. 1088-1094 ◽

Cited By ~ 1

Author(s):

Nisha Ranganathan ◽

Rebecca Johnson ◽

Andrew M. Edwards

Keyword(s):

Staphylococcus Aureus ◽

Stress Response ◽

Human Blood ◽

Chronic Infection ◽

Type Species ◽

Content Type ◽

General Stress Response ◽

Link Type ◽

General Stress ◽

Immune Defences

Staphylococcus aureus is a frequent cause of invasive human infections such as bacteraemia and infective endocarditis. These infections frequently relapse or become chronic, suggesting that the pathogen has mechanisms to tolerate the twin threats of therapeutic antibiotics and host immunity. The general stress response of S. aureus is regulated by the alternative sigma factor B (σB) and provides protection from multiple stresses including oxidative, acidic and heat. σB also contributes to virulence, intracellular persistence and chronic infection. However, the protective effect of σB on bacterial survival during exposure to antibiotics or host immune defences is poorly characterized. We found that σB promotes the survival of S. aureus exposed to the antibiotics gentamicin, ciprofloxacin, vancomycin and daptomycin, but not oxacillin or clindamycin. We also found that σB promoted staphylococcal survival in whole human blood, most likely via its contribution to oxidative stress resistance. Therefore, we conclude that the general stress response of S. aureus may contribute to the development of chronic infection by conferring tolerance to both antibiotics and host immune defences.

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Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

10.1101/2020.07.30.230003 ◽

2020 ◽

Author(s):

Madeline R. Barron ◽

Roberto J. Cieza ◽

David R. Hill ◽

Sha Huang ◽

Veda K. Yadagiri ◽

...

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

General Stress Response ◽

E Coli ◽

Germ Free ◽

General Stress ◽

Intestinal Organoids ◽

Mouse Intestine

AbstractPluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naïve intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic Escherichia coli strain, ECOR2, into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic ∆rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the ∆rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses.ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen differ from those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.

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Brucella abortus HtrA Functions as an Authentic Stress Response Protease but Is Not Required for Wild-Type Virulence in BALB/c Mice

Infection and Immunity ◽

10.1128/iai.69.9.5911-5913.2001 ◽

2001 ◽

Vol 69 (9) ◽

pp. 5911-5913 ◽

Cited By ~ 24

Author(s):

Robert W. Phillips ◽

R. Martin Roop

Keyword(s):

Stress Response ◽

Mouse Model ◽

Brucella Abortus ◽

Parental Strain ◽

Biological Function ◽

Critical Role ◽

Wild Type ◽

Secondary Line

ABSTRACT A second mutation has recently been identified in the previously described Brucella abortus htrA mutant PHE1. As a result of this finding, a new B. abortus htrAmutant, designated RWP11, was constructed to evaluate the biological function of the Brucella HtrA protease. RWP11 is more sensitive to oxidative killing in vitro and less resistant to killing by cultured murine neutrophils and macrophages than the virulent parental strain 2308 but is not attenuated in BALB/c mice through 4 weeks postinfection. The in vitro phenotype of B. abortusRWP11 is consistent with the proposed function of bacterial HtrA proteases as components of a secondary line of defense against oxidative damage. The in vivo phenotype of this mutant, however, indicates that, unlike the corresponding Salmonellaand Yersinia proteins, Brucella HtrA does not play a critical role in virulence in the mouse model.

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A Novel Piperazine-Based Drug Lead for Cryptosporidiosis from the Medicines for Malaria Venture Open-Access Malaria Box

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01505-17 ◽

2018 ◽

Vol 62 (4) ◽

pp. e01505-17 ◽

Cited By ~ 17

Author(s):

R. S. Jumani ◽

K. Bessoff ◽

M. S. Love ◽

P. Miller ◽

E. E. Stebbins ◽

...

Keyword(s):

Mouse Model ◽

Drug Development ◽

Early Stage ◽

New Drugs ◽

Cryptosporidium Hominis ◽

Content Type ◽

Knockout Mouse Model ◽

Malaria Box

ABSTRACTCryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drivein vivoefficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound forCryptosporidiumdrug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies,in vitrotoxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound againstCryptosporidium parvumIowa and field isolates was comparable to that againstCryptosporidium hominis. Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic forC. parvum, we developed a novelin vitroparasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stageCryptosporidiumdrug leads and may aid in planningin vivoefficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents.

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Role of Toxoplasma gondii Chloroquine Resistance Transporter in Bradyzoite Viability and Digestive Vacuole Maintenance

mBio ◽

10.1128/mbio.01324-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 7

Author(s):

Geetha Kannan ◽

Manlio Di Cristina ◽

Aric J. Schultz ◽

My-Hang Huynh ◽

Fengrong Wang ◽

...

Keyword(s):

Toxoplasma Gondii ◽

Chronic Infection ◽

Chloroquine Resistance ◽

Congenital Defects ◽

Functional Link ◽

Content Type ◽

Chloroquine Resistance Transporter

ABSTRACT Toxoplasma gondii is a ubiquitous pathogen that can cause encephalitis, congenital defects, and ocular disease. T. gondii has also been implicated as a risk factor for mental illness in humans. The parasite persists in the brain as slow-growing bradyzoites contained within intracellular cysts. No treatments exist to eliminate this form of parasite. Although proteolytic degradation within the parasite lysosome-like vacuolar compartment (VAC) is critical for bradyzoite viability, whether other aspects of the VAC are important for parasite persistence remains unknown. An ortholog of Plasmodium falciparum chloroquine resistance transporter (CRT), TgCRT, has previously been identified in T. gondii. To interrogate the function of TgCRT in chronic-stage bradyzoites and its role in persistence, we knocked out TgCRT in a cystogenic strain and assessed VAC size, VAC digestion of host-derived proteins and parasite autophagosomes, and the viability of in vitro and in vivo bradyzoites. We found that whereas parasites deficient in TgCRT exhibit normal digestion within the VAC, they display a markedly distended VAC and their viability is compromised both in vitro and in vivo. Interestingly, impairing VAC proteolysis in TgCRT-deficient bradyzoites restored VAC size, consistent with a role for TgCRT as a transporter of products of digestion from the VAC. In conjunction with earlier studies, our current findings suggest a functional link between TgCRT and VAC proteolysis. This study provides further evidence of a crucial role for the VAC in bradyzoite persistence and a new potential VAC target to abate chronic Toxoplasma infection. IMPORTANCE Individuals chronically infected with the intracellular parasite Toxoplasma gondii are at risk of experiencing reactivated disease that can result in progressive loss of vision. No effective treatments exist for chronic toxoplasmosis due in part to a poor understanding of the biology underlying chronic infection and a lack of well-validated potential targets. We show here that a T. gondii transporter is functionally linked to protein digestion within the parasite lysosome-like organelle and that this transporter is necessary to sustain chronic infection in culture and in experimentally infected mice. Ablating the transporter results in severe bloating of the lysosome-like organelle. Together with earlier work, this study suggests the parasite’s lysosome-like organelle is vital for parasite survival, thus rendering it a potential target for diminishing infection and reducing the risk of reactivated disease.

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Fitness, Stress Resistance, and Extraintestinal Virulence in Escherichia coli

Infection and Immunity ◽

10.1128/iai.01329-12 ◽

2013 ◽

Vol 81 (8) ◽

pp. 2733-2742 ◽

Cited By ~ 26

Author(s):

Alexandre Bleibtreu ◽

Pierre-Alexis Gros ◽

Cédric Laouénan ◽

Olivier Clermont ◽

Hervé Le Nagard ◽

...

Keyword(s):

Escherichia Coli ◽

Growth Rate ◽

Stress Response ◽

Mouse Model ◽

Stress Resistance ◽

Maximum Growth ◽

Competitive Fitness ◽

Content Type ◽

General Stress Response ◽

E Coli

ABSTRACTThe extraintestinal virulence ofEscherichia coliis dependent on numerous virulence genes. However, there is growing evidence for a role of the metabolic properties and stress responses of strains in pathogenesis. We assessed the respective roles of these factors in strain virulence by developing phenotypic assays for measuringin vitroindividual and competitive fitness and the general stress response, which we applied to 82 commensal and extraintestinal pathogenicE. colistrains previously tested in a mouse model of sepsis. Individual fitness properties, in terms of maximum growth rates in various media (Luria-Bertani broth with and without iron chelator, minimal medium supplemented with gluconate, and human urine) and competitive fitness properties, estimated as the mean relative growth rate per generation in mixed cultures with a reference fluorescentE. colistrain, were highly diverse between strains. The activity of the main general stress response regulator, RpoS, as determined by iodine staining of the colonies, H2O2resistance, andrpoSsequencing, was also highly variable. No correlation between strain fitness and stress resistance and virulence in the mouse model was found, except that the maximum growth rate in urine was higher for virulent strains. Multivariate analysis showed that the number of virulence factors was the only independent factor explaining the virulence in mice. At the species level, growth capacity and stress resistance are heterogeneous properties that do not contribute significantly to the intrinsic virulence of the strains.

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Exposure of Bacillus subtilis to Low Pressure (5 Kilopascals) Induces Several Global Regulons, Including Those Involved in the SigB-Mediated General Stress Response

Applied and Environmental Microbiology ◽

10.1128/aem.00885-14 ◽

2014 ◽

Vol 80 (16) ◽

pp. 4788-4794 ◽

Cited By ~ 12

Author(s):

Samantha M. Waters ◽

José A. Robles-Martínez ◽

Wayne L. Nicholson

Keyword(s):

Bacillus Subtilis ◽

Stress Response ◽

High Pressures ◽

Low Pressure ◽

Bacterial Cells ◽

Content Type ◽

General Stress Response ◽

Cellular Processes ◽

General Stress ◽

Microbial Responses

ABSTRACTStudies of how microorganisms respond to pressure have been limited mostly to the extreme high pressures of the deep sea (i.e., the piezosphere). In contrast, despite the fact that the growth of most bacteria is inhibited at pressures below ∼2.5 kPa, little is known of microbial responses to low pressure (LP). To study the global LP response, we performed transcription microarrays onBacillus subtiliscells grown under normal atmospheric pressure (∼101 kPa) and a nearly inhibitory LP (5 kPa), equivalent to the pressure found at an altitude of ∼20 km. Microarray analysis revealed altered levels of 363 transcripts belonging to several global regulons (AbrB, CcpA, CodY, Fur, IolR, ResD, Rok, SigH, Spo0A). Notably, the highest number of upregulated genes, 86, belonged to the SigB-mediated general stress response (GSR) regulon. Upregulation of the GSR by LP was confirmed by monitoring the expression of the SigB-dependentctc-lacZreporter fusion. Measuring transcriptome changes resulting from exposure of bacterial cells to LP reveals insights into cellular processes that may respond to LP exposure.

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Two-Tiered Histidine Kinase Pathway Involved in Heat Shock and Salt Sensing in the General Stress Response of Sphingomonas melonis Fr1

Journal of Bacteriology ◽

10.1128/jb.00019-15 ◽

2015 ◽

Vol 197 (8) ◽

pp. 1466-1477 ◽

Cited By ~ 12

Author(s):

Andreas Kaczmarczyk ◽

Ramon Hochstrasser ◽

Julia A. Vorholt ◽

Anne Francez-Charlot

Keyword(s):

Stress Response ◽

Heat Shock ◽

Experimental Evidence ◽

Histidine Kinase ◽

Response Regulators ◽

Catalytic Core ◽

Content Type ◽

General Stress Response ◽

General Stress ◽

Kinase Pathway

ABSTRACTThe general stress response (GSR) allows bacteria to monitor and defend against a broad set of unrelated, adverse environmental conditions. InAlphaproteobacteria, the key step in GSR activation is phosphorylation of the response regulator PhyR. InSphingomonas melonisFr1, seven PhyR-activating kinases (Paks), PakA to PakG, are thought to directly phosphorylate PhyR under different stress conditions, but the nature of the activating signals remains obscure. PakF, a major sensor of NaCl and heat shock, lacks a putative sensor domain but instead harbors a single receiver (REC) domain (PakFREC) N-terminal to its kinase catalytic core. Such kinases are called “hybrid response regulators” (HRRs). How HRRs are able to perceive signals in the absence of a true sensor domain has remained largely unexplored. In the present work, we show that stresses are actually sensed by another kinase, KipF (kinase of PakF), which phosphorylates PakFRECand thereby activates PakF. KipF is a predicted transmembrane kinase, harboring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic kinase catalytic core. We demonstrate that KipF senses different salts through its CHASE3 domain but is not a sensor of general osmotic stress. While salt sensing depends on the CHASE3 domain, heat shock sensing does not, suggesting that these stresses are perceived by different mechanisms. In summary, our results establish a two-tiered histidine kinase pathway involved in activation of the GSR inS. melonisFr1 and provide the first experimental evidence for the so far uncharacterized CHASE3 domain as a salt sensor.IMPORTANCEHybrid response regulators (HRRs) represent a particular class of histidine kinases harboring an N-terminal receiver (REC) domain instead of a true sensor domain. This suggests that the actual input for HRRs may be phosphorylation of the REC domain. In the present study, we addressed this question by using the HRR PakF. Our results suggest that PakF is activated through phosphorylation of its REC domain and that this is achieved by another kinase, KipF. KipF senses heat shock and salt stress, with the latter requiring the periplasmic CHASE3 domain. This work not only suggests that HRRs work in two-tiered histidine kinase pathways but also provides the first experimental evidence for a role of the so far uncharacterized CHASE3 domain in salt sensing.

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Filamentous Bacteriophage Produced by Pseudomonas aeruginosa Alters the Inflammatory Response and Promotes Noninvasive Infection In Vivo

Infection and Immunity ◽

10.1128/iai.00648-16 ◽

2016 ◽

Vol 85 (1) ◽

Cited By ~ 36

Author(s):

Patrick R. Secor ◽

Lia A. Michaels ◽

Kate S. Smigiel ◽

Maryam G. Rohani ◽

Laura K. Jennings ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Inflammatory Response ◽

Chronic Infection ◽

Bacterial Invasion ◽

Airway Epithelial ◽

Content Type ◽

Epithelial Cultures ◽

Opportunistic Human Pathogen

ABSTRACT Pseudomonas aeruginosa is an important opportunistic human pathogen that lives in biofilm-like cell aggregates at sites of chronic infection, such as those that occur in the lungs of patients with cystic fibrosis and nonhealing ulcers. During growth in a biofilm, P. aeruginosa dramatically increases the production of filamentous Pf bacteriophage (Pf phage). Previous work indicated that when in vivo Pf phage production was inhibited, P. aeruginosa was less virulent. However, it is not clear how the production of abundant quantities of Pf phage similar to those produced by biofilms under in vitro conditions affects pathogenesis. Here, using a murine pneumonia model, we show that the production of biofilm-relevant amounts of Pf phage prevents the dissemination of P. aeruginosa from the lung. Furthermore, filamentous phage promoted bacterial adhesion to mucin and inhibited bacterial invasion of airway epithelial cultures, suggesting that Pf phage traps P. aeruginosa within the lung. The in vivo production of Pf phage was also associated with reduced lung injury, reduced neutrophil recruitment, and lower cytokine levels. Additionally, when producing Pf phage, P. aeruginosa was less prone to phagocytosis by macrophages than bacteria not producing Pf phage. Collectively, these data suggest that filamentous Pf phage alters the progression of the inflammatory response and promotes phenotypes typically associated with chronic infection.

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EGR3-HDAC6-IL-27 Axis Mediates Allergic Inflammation and Is Necessary for Tumorigenic Potential of Cancer Cells Enhanced by Allergic Inflammation-Promoted Cellular Interactions

Frontiers in Immunology ◽

10.3389/fimmu.2021.680441 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yoojung Kwon ◽

Misun Kim ◽

Youngmi Kim ◽

Myeong Seon Jeong ◽

Hyun Suk Jung ◽

...

Keyword(s):

Mouse Model ◽

Binding Site ◽

Allergic Inflammation ◽

Metastatic Potential ◽

Cellular Interactions ◽

Promoter Sequences ◽

Potential Binding ◽

Potential Binding Site

The objective of this study was to investigate mechanisms of allergic inflammation both in vitro and in vivo in details. For this, RNA sequencing was performed. Early growth response 3 gene (Egr3) was one of the most highly upregulated genes in rat basophilic leukemia (RBL2H3) cells stimulated by antigen. The role of Egr3 in allergic inflammation has not been studied extensively. Egr3 was necessary for passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis (PSA). Egr3 promoter sequences contained potential binding site for NF-κB p65. NF-κB p65 directly regulated Egr3 expression and mediated allergic inflammation in vitro. Histone deacetylases (HDACs) is known to be involved in allergic airway inflammation. HDAC6 promoter sequences contained potential binding site for EGR3. EGR3 showed binding to promoter sequences of HDAC6. EGR3 was necessary for increased expression of histone deacetylase 6 (HDAC6) in antigen-stimulated RBL2H3 cells. HDAC6 mediated allergic inflammation in vitro and PSA. TargetScan analysis predicted that miR-182-5p was a negative regulator of EGR3. Luciferase activity assay confirmed that miR-182-5p was a direct regulator of EGR3. MiR-182-5p mimic inhibited allergic inflammation both in vitro and in vivo. Cytokine array showed that HDAC6 was necessary for increased interleukin-27 (IL-27) expression in BALB/C mouse model of PSA. Antigen stimulation did not affect expression of EBI3, another subunit of IL-27 in RBL2H3 cells or BALB/C mouse model of PCA or PSA. IL-27 receptor alpha was shown to be able to bind to HDAC6. IL-27 p28 mediated allergic inflammation in vitro, PCA, and PSA. Mouse recombinant IL-27 protein promoted features of allergic inflammation in an antigen-independent manner. HDAC6 was necessary for tumorigenic and metastatic potential enhanced by PSA. PSA enhanced the metastatic potential of mouse melanoma B16F1 cells in an IL-27-dependent manner. Experiments employing culture medium and mouse recombinant IL-27 protein showed that IL-27 mediated and promoted cellular interactions involving B16F1 cells, lung macrophages, and mast cells during allergic inflammation. IL-27 was present in exosomes of antigen-stimulated RBL2H3 cells. Exosomes from antigen-stimulated RBL2H3 cells enhanced invasion of B16F1 melanoma cells in an IL-27-dependemt manner. These results present evidence that EGR3-HDAC6-IL-27 axis can regulate allergic inflammation by mediating cellular interactions.

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