Simulated colonic fluid replicates the in vivo growth capabilities of Citrobacter rodentium mutants and highlights a critical role for the Cpx envelope stress response in mediating stressors encountered in the gastrointestinal tract

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. While in the host, C. rodentium must adapt to stresses in the gastrointestinal tract such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression and is necessary for C. rodentium pathogenesis in mice. To investigate genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, RNA-Seq, SILAC, and microarray data from previous research was mined and the genes yebE, ygiB, bssR, and htpX were confirmed to be strongly upregulated by the presence of CpxRA using lux reporter constructs. To determine the function of these genes in vivo, knockout mutants were tested in C57Bl/6J and C3H/HeJ mice. Although none of the mutants exhibited marked virulence phenotypes, the ΔcpxRA mutant had reduced colonization and attenuated virulence, as previously determined. We also found that the absence of the Cpx ESR resulted in higher expression of the LEE master regulator, ler. In addition, we determined that the ΔcpxRA mutant had a growth defect in medium simulating the colon, as did several of the mutants bearing deletions in Cpx-upregulated genes. Overall, these results indicate that the ΔcpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

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Phyletic distribution and diversification of the Phage Shock Protein stress response system in bacteria and archaea

10.1101/2021.02.15.431232 ◽

2021 ◽

Author(s):

Philipp F. Popp ◽

Vadim M. Gumerov ◽

Ekaterina P. Andrianova ◽

Lisa Bewersdorf ◽

Thorsten Mascher ◽

...

Keyword(s):

Stress Response ◽

Large Scale ◽

Cell Envelope ◽

Model Organisms ◽

Microbial World ◽

Natural Classification ◽

Response System ◽

Core Proteins ◽

Envelope Stress

AbstractThe bacterial cell envelope is an essential structure that protects the cell from environmental threats, while simultaneously serving as communication interface and diffusion barrier. Therefore, maintaining cell envelope integrity is of vital importance for all microorganisms. Not surprisingly, evolution has shaped conserved protection networks that connect stress perception, transmembrane signal transduction and mediation of cellular responses upon cell envelope stress. The phage shock protein (PSP) stress response is one of such conserved protection networks. Most of the knowledge about the Psp response comes from studies in the Gram-negative model bacterium, Escherichia coli where the Psp system consists of several well-defined protein components. Homologous systems were identified in representatives of Proteobacteria, Actinobacteria, and Firmicutes; however, the Psp system distribution in the microbial world remains largely unknown. By carrying out a large-scale, unbiased comparative genomics analysis, we found components of the Psp system in many bacterial and archaeal phyla and demonstrated that the PSP system deviates dramatically from the proteobacterial prototype. Two of its core proteins, PspA and PspC, have been integrated in various (often phylum-specifically) conserved protein networks during evolution. Based on protein sequence and gene neighborhood analyses of pspA and pspC homologs, we built a natural classification system of PSP networks in bacteria and archaea. We performed a comprehensive in vivo protein interaction screen for the PSP network newly identified in the Gram-positive model organism Bacillus subtilis and found a strong interconnected PSP response system, illustrating the validity of our approach. Our study highlights the diversity of PSP organization and function across many bacterial and archaeal phyla and will serve as foundation for future studies of this envelope stress response beyond model organisms.

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The CpxQ sRNA Negatively Regulates Skp To Prevent Mistargeting of β-Barrel Outer Membrane Proteins into the Cytoplasmic Membrane

mBio ◽

10.1128/mbio.00312-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 27

Author(s):

Marcin Grabowicz ◽

Daria Koren ◽

Thomas J. Silhavy

Keyword(s):

Stress Response ◽

Membrane Proteins ◽

Outer Membrane ◽

Protein Misfolding ◽

Outer Membrane Proteins ◽

Inner Membrane ◽

Bam Complex ◽

Envelope Stress ◽

Periplasmic Chaperone

ABSTRACT The promoter most strongly induced upon activation of the Cpx two-component envelope stress response is the cpxP promoter. The 3′ untranscribed region (UTR) of the cpxP transcript is shown to produce a small RNA (sRNA), CpxQ. We investigated the role of CpxQ in combating envelope stress. Remarkably, the two effectors specified by the transcript are deployed to combat distinct stresses in different cellular compartments. CpxP acts in both a regulatory negative-feedback loop and as an effector that combats periplasmic protein misfolding. We find that CpxQ combats toxicity at the inner membrane (IM) by downregulating the synthesis of the periplasmic chaperone Skp. Our data indicate that this regulation prevents Skp from inserting β-barrel outer membrane proteins (OMPs) into the IM, a lethal event that likely collapses the proton motive force. Our findings suggest that Skp can fold and directly insert OMPs into a lipid bilayer in vivo without the aid of the Bam complex. IMPORTANCE Skp is a well-characterized periplasmic chaperone that binds unfolded OMPs. Surprisingly, we find that Skp can catalyze the folding and mistargeting of OMPs into the inner membrane without the aid of the other cellular proteins that normally assemble OMPs. Several OMPs function as diffusion pores. Accordingly, their mistargeting is lethal because it depolarizes the inner membrane. We show that the most highly expressed transcript of the Cpx stress response produces an sRNA from the 3′ UTR, CpxQ, which combats this potential toxicity by downregulating Skp production. Defects in OMP assembly trigger the σ E response to upregulate factors, including Skp, that promote OMP folding. The Cpx response downregulates σ E . Our findings reveal that this heretofore puzzling hierarchy exists to protect the inner membrane.

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Identification of a novel, NF-κB nucleolar stress response pathway

10.1101/100255 ◽

2017 ◽

Author(s):

Jingyu Chen ◽

Ian T Lobb ◽

Pierre Morin ◽

Sonia M Novo ◽

James Simpson ◽

...

Keyword(s):

Stress Response ◽

Ex Vivo ◽

Critical Role ◽

Rdna Transcription ◽

Therapeutic Implications ◽

Stress Effects ◽

Nucleolar Stress ◽

Stress Response Pathway ◽

Cdk4 Inhibition

Abstractp53 as an effector of nucleolar stress is well defined, but p53 independent mechanisms are largely unknown. Like p53, the NF-κB transcription factor plays a critical role in maintaining cellular homeostasis under stress. Many stresses that stimulate NF-κB also disrupt nucleoli. However, the link between nucleolar function and activation of the NF-κB pathway is as yet unknown. Here we demonstrate that siRNA silencing of PolI complex components stimulates NF-κB signalling. Unlike p53 nucleolar stress response, this effect does not appear to be linked to inhibition of rDNA transcription. We show that specific stress stimuli of NF-κB induce degradation of a critical component of the PolI complex, TIF-IA. This degradation precedes activation of the NF-κB pathway and is associated with an atypical nucleolar architecture. It is mimicked by CDK4 inhibition and is dependent upon upstream binding factor (UBF) and p14ARF. We show that blocking stress effects on TIF-IA blocks their ability to activate the NF-κB pathway. Finally, usingex vivoculture, we show a strong correlation between degradation of TIF-IA and activation of NF-κB in freshly resected, human colorectal tumours exposed to the chemopreventative agent, aspirin. Together, our study provides compelling evidence for a new, NF-κB nucleolar stress response pathway that has in vivo relevance and therapeutic implications.

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Alternative Sigma Factor RpoE Is Important for Vibrio parahaemolyticus Cell Envelope Stress Response and Intestinal Colonization

Infection and Immunity ◽

10.1128/iai.01854-14 ◽

2014 ◽

Vol 82 (9) ◽

pp. 3667-3677 ◽

Cited By ~ 20

Author(s):

Brandy Haines-Menges ◽

W. Brian Whitaker ◽

E. Fidelma Boyd

Keyword(s):

Stress Response ◽

Vibrio Parahaemolyticus ◽

Response Regulator ◽

Cell Envelope ◽

Polymyxin B ◽

Content Type ◽

Alternative Sigma Factors ◽

Envelope Stress

ABSTRACTVibrio parahaemolyticusis a halophile that inhabits brackish waters and a wide range of hosts, including crustaceans, fish, mollusks, and humans. In humans, it is the leading cause of bacterial seafood-borne gastroenteritis. The focus of this work was to determine the role of alternative sigma factors in the stress response ofV. parahaemolyticusRIMD2210633, an O3:K6 pandemic isolate. Bioinformatics identified five putative extracytoplasmic function (ECF) family of alternative sigma factors: VP0055, VP2210, VP2358, VP2578, and VPA1690. ECF factors typically respond to cell wall/cell envelope stress, iron levels, and the oxidation state of the cell. We have demonstrated here that one such sigma factor, VP2578, a homologue of RpoE fromEscherichia coli, is important for survival under a number of cell envelope stress conditions and in gastrointestinal colonization of a streptomycin-treated adult mouse. In this study, we determined that anrpoEdeletion mutant strain BHM2578 compared to the wild type (WT) was significantly more sensitive to polymyxin B, ethanol, and high-temperature stresses. We demonstrated that inin vivocompetition assays between therpoEmutant and the WT marked with the β-galactosidase genelacZ(WBWlacZ), the mutant strain was defective in colonization compared to the WT. In contrast, deletion of therpoSstress response regulator did not affectin vivosurvival. In addition, we examined the role of the outer membrane protein, OmpU, which inV. choleraeis proposed to be the sole activator of RpoE. We found that anompUdeletion mutant was sensitive to bile salt stress but resistant to polymyxin B stress, indicating OmpU is not essential for the cell envelope stress responses or RpoE function. Overall, these data demonstrate that RpoE is a key cell envelope stress response regulator and, similar toE. coli, RpoE may have several factors that stimulate its function.

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Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.00430-16 ◽

2016 ◽

Vol 84 (11) ◽

pp. 3141-3151 ◽

Cited By ~ 7

Author(s):

Adrienne C. Showman ◽

George Aranjuez ◽

Philip P. Adams ◽

Mollie W. Jewett

Keyword(s):

Oxidative Stress ◽

Stress Response ◽

Borrelia Burgdorferi ◽

Molecular Mechanisms ◽

De Novo ◽

Oxidative Stress Response ◽

Growth Defect ◽

Content Type

A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo , we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δ bb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 . In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.

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Transcription Factor MEF2D is Required for the Maintenance of MLL-rearranged Acute Myeloid Leukemia

Blood Advances ◽

10.1182/bloodadvances.2021004469 ◽

2021 ◽

Author(s):

Lianzhong Zhao ◽

Pengcheng Zhang ◽

Phillip M Galbo ◽

Xinyue Zhou ◽

Sajesan Aryal ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Transcription Factor ◽

Myeloid Leukemia ◽

Critical Role ◽

Leukemia Cell ◽

Myeloid Differentiation ◽

Growth Defect ◽

Strong Negative Correlation ◽

Acute Myeloid

Acute myeloid leukemia (AML) with MLL-rearrangement (MLL-r) comprises approximately 10% of all AML cases and portends poor outcomes. Much remains uncovered on how MLL-r AML drives leukemia development while preventing cells from normal myeloid differentiation. Here, we identified that transcription factor MEF2D is a super-enhancer-associated, highly expressed gene in MLL-r AML. Knockout of MEF2D profoundly impaired leukemia growth, induced myeloid differentiation, and delayed oncogenic progression in vivo. Mechanistically, MEF2D loss led to robust activation of a CEBPE-centered myeloid differentiation program in AML cells. Chromatin profiling revealed that MEF2D binds to and suppresses the chromatin accessibility of CEBPE cis-regulatory regions. In human acute leukemia samples, MEF2D expression showed a strong negative correlation with the expression of CEBPE. Depletion of CEBPE partially rescued the cell growth defect and myeloid cell differentiation induced by the loss of MEF2D. Lastly, we show that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced anti-leukemia effects. Collectively, our findings suggest that MEF2D plays a critical role in human MLL-r AML and uncover the MEF2D-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block.

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Mutations That Impact the Enteropathogenic Escherichia coli Cpx Envelope Stress Response Attenuate Virulence in Galleria mellonella

Infection and Immunity ◽

10.1128/iai.00081-12 ◽

2012 ◽

Vol 80 (9) ◽

pp. 3077-3085 ◽

Cited By ~ 40

Author(s):

S. Leuko ◽

T. L. Raivio

Keyword(s):

Escherichia Coli ◽

Stress Response ◽

Type Iii Secretion ◽

Galleria Mellonella ◽

Infection Model ◽

Virulence Determinants ◽

Content Type ◽

Type Iii ◽

Envelope Stress

ABSTRACTIn this paper, we show that the larvae of the greater wax moth,Galleria mellonella, can be used as a model to study enteropathogenicEscherichia coli(EPEC) virulence.G. mellonellalarvae are killed after infection with EPEC type strain E2348/69 but not by an attenuated derivative that expresses diminished levels of the major virulence determinants or by a mutant specifically defective in type III secretion (T3S). Infecting EPEC inhabit the larval hemocoel only briefly and then become localized to melanized capsules, where they remain extracellular. Previously, it was shown that mutations affecting the Cpx envelope stress response lead to diminished expression of the bundle-forming pilus (BFP) and the type III secretion system (T3SS). We demonstrate that mutations that activate the Cpx pathway have a dramatic effect on the ability of the bacterium to establish a lethal infection, and this is correlated with an inability to growin vivo. Infection with allE. colistrains led to increased expression of the antimicrobial peptides (AMPs) gloverin and cecropin, although strain- and AMP-specific differences were observed, suggesting that theG. mellonellahost perceives attenuated strains and Cpx mutants in unique manners. Overall, this study shows thatG. mellonellais an economical, alternative infection model for the preliminary study of EPEC host-pathogen interactions, and that induction of the Cpx envelope stress response leads to defects in virulence.

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Contribution of Citrulline Ureidase to Francisella tularensis Strain Schu S4 Pathogenesis

Journal of Bacteriology ◽

10.1128/jb.00212-09 ◽

2009 ◽

Vol 191 (15) ◽

pp. 4798-4806 ◽

Cited By ~ 14

Author(s):

Manish Mahawar ◽

Girish S. Kirimanjeswara ◽

Dennis W. Metzger ◽

Chandra Shekhar Bakshi

Keyword(s):

Francisella Tularensis ◽

Growth Conditions ◽

Growth Defect ◽

Bacterial Burden ◽

Time To Death ◽

Virulent Strains ◽

Absolute Requirement ◽

In Vivo Growth ◽

Schu S4

ABSTRACT The citrulline ureidase (CTU) activity has been shown to be associated with highly virulent Francisella tularensis strains, including Schu S4, while it is absent in avirulent or less virulent strains. A definitive role of the ctu gene in virulence and pathogenesis of F. tularensis Schu S4 has not been assessed; thus, an understanding of the significance of this phenotype is long overdue. CTU is a carbon-nitrogen hydrolase encoded by the citrulline ureidase (ctu) gene (FTT0435) on the F. tularensis Schu S4 genome. In the present study, we evaluated the contribution of the ctu gene in the virulence of category A agent F. tularensis Schu S4 by generating a nonpolar deletion mutant, the Δctu mutant. The deletion of the ctu gene resulted in loss of CTU activity, which was restored by transcomplementing the ctu gene. The Δctu mutant did not exhibit any growth defect under acellular growth conditions; however, it was impaired for intramacrophage growth in resting as well as gamma interferon-stimulated macrophages. The Δctu mutant was further tested for its virulence attributes in a mouse model of respiratory tularemia. Mice infected intranasally with the Δctu mutant showed significantly reduced bacterial burden in the lungs, liver, and spleen compared to wild-type (WT) Schu S4-infected mice. The reduced bacterial burden in mice infected with the Δctu mutant was also associated with significantly lower histopathological scores in the lungs. Mice infected with the Δctu mutant succumbed to infection, but they survived longer and showed significantly extended median time to death compared to that shown by WT Schu S4-infected mice. To conclude, this study demonstrates that ctu contributes to intracellular survival, in vivo growth, and pathogenesis. However, ctu is not an absolute requirement for the virulence of F. tularensis Schu S4 in mice.

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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 Continuous Battle for Host-Derived Glycans Between a Mucus Specialist and a Glycan Generalist in vitro and in vivo

Frontiers in Microbiology ◽

10.3389/fmicb.2021.632454 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ioannis Kostopoulos ◽

Steven Aalvink ◽

Petia Kovatcheva-Datchary ◽

Bart Nijsse ◽

Fredrik Bäckhed ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Lipid A ◽

Environmental Changes ◽

Membrane Attack Complex ◽

Glycoside Hydrolases ◽

Gene Encoding ◽

Macpf Domain ◽

Upregulated Genes

The human gastrointestinal tract is colonized by a diverse microbial community, which plays a crucial role in human health. In the gut, a protective mucus layer that consists of glycan structures separates the bacteria from the host epithelial cells. These host-derived glycans are utilized by bacteria that have adapted to this specific compound in the gastrointestinal tract. Our study investigated the close interaction between two distinct gut microbiota members known to use mucus glycans, the generalist Bacteroides thetaiotaomicron and the specialist Akkermansia muciniphila in vitro and in vivo. The in vitro study, in which mucin was the only nutrient source, indicated that B. thetaiotaomicron significantly upregulated genes coding for Glycoside Hydrolases (GHs) and mucin degradation activity when cultured in the presence of A. muciniphila. Furthermore, B. thetaiotaomicron significantly upregulated the expression of a gene encoding for membrane attack complex/perforin (MACPF) domain in co-culture. The transcriptome analysis also indicated that A. muciniphila was less affected by the environmental changes and was able to sustain its abundance in the presence of B. thetaiotaomicron while increasing the expression of LPS core biosynthesis activity encoding genes (O-antigen ligase, Lipid A and Glycosyl transferases) as well as ABC transporters. Using germ-free mice colonized with B. thetaiotaomicron and/or A. muciniphila, we observed a more general glycan degrading profile in B. thetaiotaomicron while the expression profile of A. muciniphila was not significantly affected when colonizing together, indicating that two different nutritional niches were established in mice gut. Thus, our results indicate that a mucin degrading generalist adapts to its changing environment, depending on available carbohydrates while a mucin degrading specialist adapts by coping with competing microorganism through upregulation of defense related genes.

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