scholarly journals Phosphate transporter PstSCAB of Campylobacter jejuni is essential for lactate-dependent growth and colonization in chickens

2019 ◽  
Author(s):  
Ritam Sinha ◽  
Rhiannon M. LeVeque ◽  
Marvin Q. Bowlin ◽  
Michael J. Gray ◽  
Victor J. DiRita
Keyword(s):  
Stationary Phase ◽  
Campylobacter Jejuni ◽  
Phosphate Transporter ◽  
Multi Drug Resistance ◽  
Wild Type ◽  
Gastrointestinal Infections ◽  
Poor Growth ◽  
High Affinity ◽  
Polyphosphate Metabolism

AbstractCampylobacter jejuni causes acute gastroenteritis world-wide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous RNASeq experiments showed that transcripts from an operon encoding a high affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNASeq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our experimental results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. C. jejuni were highly attenuated in colonization of newly hatched chicks, recovered at levels several orders of magnitude below wild type. Mutant and wild type grew similarly in complex media but the pstSCAB mutant exhibited a significant growth defect in minimal media supplemented with L-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival.ImportanceC. jejuni causes millions of gastrointestinal infections annually worldwide. Poultry and poultry products are major sources of C. jejuni infection to human as the microbe is a commensal colonizer of the chicken gastrointestinal tract. Due to the emergence of multi-drug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB were highly expressed during colonization of C. jejuni in chicken and human. In this study, we address the role this high-affinity phosphate transporter PstSCAB of C. jejuni on chicken colonization and for its general physiology. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses and during growth on lactate, a potential substrate for growth of C. jejuni in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms to control the bacterial burden in both chicken and human.

scholarly journals Phosphate Transporter PstSCAB of Campylobacter jejuni Is a Critical Determinant of Lactate-Dependent Growth and Colonization in Chickens

2020 ◽  
Vol 202 (7) ◽  
Author(s):  
Ritam Sinha ◽  
Rhiannon M. LeVeque ◽  
Marvin Q. Bowlin ◽  
Michael J. Gray ◽  
Victor J. DiRita
Keyword(s):  
Stationary Phase ◽  
Campylobacter Jejuni ◽  
Phosphate Transporter ◽  
Rna Seq ◽  
Wild Type ◽  
Content Type ◽  
High Affinity ◽  
Polyphosphate Metabolism

ABSTRACT Campylobacter jejuni causes acute gastroenteritis worldwide and is transmitted primarily through poultry, in which it is often a commensal member of the intestinal microbiota. Previous transcriptome sequencing (RNA-Seq) experiment showed that transcripts from an operon encoding a high-affinity phosphate transporter (PstSCAB) of C. jejuni were among the most abundant when the bacterium was grown in chickens. Elevated levels of the pstSCAB mRNA were also identified in an RNA-Seq experiment from human infection studies. In this study, we explore the role of PstSCAB in the biology and colonization potential of C. jejuni. Our results demonstrate that cells lacking PstSCAB survive poorly in stationary phase, in nutrient-limiting media, and under osmotic conditions reflective of those in the chicken. Polyphosphate levels in the mutant cells were elevated at stationary phase, consistent with alterations in expression of polyphosphate metabolism genes. The mutant strain was highly attenuated for colonization of newly hatched chicks, with levels of bacteria at several orders of magnitude below wild-type levels. Mutant and wild type grew similarly in complex media, but the pstS::kan mutant exhibited a significant growth defect in minimal medium supplemented with l-lactate, postulated as a carbon source in vivo. Poor growth in lactate correlated with diminished expression of acetogenesis pathway genes previously demonstrated as important for colonizing chickens. The phosphate transport system is thus essential for diverse aspects of C. jejuni physiology and in vivo fitness and survival. IMPORTANCE Campylobacter jejuni causes millions of human gastrointestinal infections annually, with poultry a major source of infection. Due to the emergence of multidrug resistance in C. jejuni, there is need to identify alternative ways to control this pathogen. Genes encoding the high-affinity phosphate transporter PstSCAB are highly expressed by C. jejuni in chickens and humans. In this study, we address the role of PstSCAB on chicken colonization and other C. jejuni phenotypes. PstSCAB is required for colonization in chicken, metabolism and survival under different stress responses, and during growth on lactate, a potential growth substrate in chickens. Our study highlights that PstSCAB may be an effective target to develop mechanisms for controlling bacterial burden in both chicken and human.

scholarly journals Effects of Combination of Different −10 Hexamers and Downstream Sequences on Stationary-Phase-Specific Sigma Factor ςS-Dependent Transcription in Pseudomonas putida

2000 ◽  
Vol 182 (23) ◽  
pp. 6707-6713 ◽  
Author(s):  
Eve-Ly Ojangu ◽  
Andres Tover ◽  
Riho Teras ◽  
Maia Kivisaar
Keyword(s):  
Stationary Phase ◽  
Pseudomonas Putida ◽  
Sigma Factor ◽  
Sigma Factors ◽  
Deficient Mutant ◽  
Wild Type ◽  
In Vivo Experiments ◽  
Silent Genes ◽  
Rna Polymerase Holoenzyme

ABSTRACT The main sigma factor activating gene expression, necessary in stationary phase and under stress conditions, is ςS. In contrast to other minor sigma factors, RNA polymerase holoenzyme containing ςS (EςS) recognizes a number of promoters which are also recognized by that containing ς70 (Eς70). We have previously shown that transposon Tn4652 can activate silent genes in starvingPseudomonas putida cells by creating fusion promoters during transposition. The sequence of the fusion promoters is similar to the ς70-specific promoter consensus. The −10 hexameric sequence and the sequence downstream from the −10 element differ among these promoters. We found that transcription from the fusion promoters is stationary phase specific. Based on in vivo experiments carried out with wild-type and rpoS-deficient mutant P. putida, the effect of ςS on transcription from the fusion promoters was established only in some of these promoters. The importance of the sequence of the −10 hexamer has been pointed out in several published papers, but there is no information about whether the sequences downstream from the −10 element can affect ςS-dependent transcription. Combination of the −10 hexameric sequences and downstream sequences of different fusion promoters revealed that ςS-specific transcription from these promoters is not determined by the −10 hexameric sequence only. The results obtained in this study indicate that the sequence of the −10 element influences ςS-specific transcription in concert with the sequence downstream from the −10 box.

scholarly journals GapA and CrmA Coexpression Is Essential for Mycoplasma gallisepticum Cytadherence and Virulence

2002 ◽  
Vol 70 (12) ◽  
pp. 6839-6845 ◽  
Author(s):  
L. Papazisi ◽  
S. Frasca ◽  
M. Gladd ◽  
X. Liao ◽  
D. Yogev ◽  
...  
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Sequence Homology ◽  
Essential Role ◽  
Mycoplasma Gallisepticum ◽  
Wild Type ◽  
Related Molecule ◽  
High Affinity ◽  
Virulence Phenotype

ABSTRACT It was previously demonstrated that avirulent Mycoplasma gallisepticum strain Rhigh (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain Rlow (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of Rhigh with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of Rhigh with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in Rhigh and GT5. In contrast, complementation of Rhigh with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type Rlow. In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis.

scholarly journals The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

2009 ◽  
Vol 191 (16) ◽  
pp. 5293-5300 ◽  
Author(s):  
Rebecca A. Weingarten ◽  
Michael E. Taveirne ◽  
Jonathan W. Olson
Keyword(s):  
Organic Acids ◽  
Succinate Dehydrogenase ◽  
Campylobacter Jejuni ◽  
Growth Phase ◽  
Tca Cycle ◽  
Fumarate Reductase ◽  
Wild Type ◽  
Succinate Dehydrogenase Activity

ABSTRACT Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat + strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat + strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat + strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat + strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat + strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat + strain colonized chickens at wild-type levels.

scholarly journals Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

2014 ◽  
Vol 197 (5) ◽  
pp. 1002-1011 ◽  
Author(s):  
Annika Flint ◽  
Alain Stintzi
Keyword(s):  
Escherichia Coli ◽  
Campylobacter Jejuni ◽  
Biochemical Properties ◽  
Wild Type ◽  
Visible Spectroscopy ◽  
Content Type ◽  
Increased Sensitivity ◽  
Uv Visible

Catalase enzymes detoxify H2O2by the dismutation of H2O2into O2and H2O through the use of hemin cofactors. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content inCampylobacter jejunicatalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol194:334–345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interactin vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed inEscherichia coli. The importance of tyrosine 57 in hemin traffickingin vivowas confirmed by introducing thecj1386Y57Aallele into aC. jejuniΔcj1386mutant background. Thecj1386Y57Amutation resulted in increased sensitivity toward H2O2relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the Δcj1386+cj1386Y57Amutant had significantly reduced hemin content compared to that of thecj1386WTbackground. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.

scholarly journals An N-Terminally Truncated RpoS (σS) Protein in Escherichia coli Is Active In Vivo and Exhibits Normal Environmental Regulation Even in the Absence of rpoS Transcriptional and Translational Control Signals

2002 ◽  
Vol 184 (12) ◽  
pp. 3167-3175 ◽  
Author(s):  
K. Rajkumari ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Stationary Phase ◽  
Translational Control ◽  
Sigma Factor ◽  
Receptor Protein ◽  
Wild Type ◽  
Truncated Protein

ABSTRACT RpoS (σS) in Escherichia coli is a stationary-phase-specific primary sigma factor of RNA polymerase which is 330 amino acids long and belongs to the eubacterial σ70 family of proteins. Conserved domain 1.1 at the N-terminal end of σ70 has been shown to be essential for RNA polymerase function, and its deletion has been shown to result in a dominant-lethal phenotype. We now report that a σS variant with a deletion of its N-terminal 50 amino acids (σSΔ1-50), when expressed in vivo either from a chromosomal rpoS::IS10 allele (in rho mutant strains) or from a plasmid-borne arabinose-inducible promoter, is as proficient as the wild type in directing transcription from the proU P1 promoter; at three other σS-dependent promoters that were tested (osmY, katE, and csiD), the truncated protein exhibited a three- to sevenfold reduced range of activities. Catabolite repression at the csiD promoter (which requires both σS and cyclic AMP [cAMP]-cAMP receptor protein for its activity) was also preserved in the strain expressing σSΔ1-50. The intracellular content of σSΔ1-50 was regulated by culture variables such as growth phase, osmolarity, and temperature in the same manner as that described earlier for σS, even when the truncated protein was expressed from a template that possessed neither the transcriptional nor the translational control elements of wild-type rpoS. Our results indicate that, unlike that in σ70, the N-terminal domain in σS may not be essential for the protein to function as a sigma factor in vivo. Furthermore, our results suggest that the induction of σS-specific promoters in stationary phase and during growth under conditions of high osmolarity or low temperature is mediated primarily through the regulation of σS protein degradation.

scholarly journals Engineered SIRPα Variants as Immunotherapeutic Adjuvants to Anticancer Antibodies

Science ◽  
2013 ◽  
Vol 341 (6141) ◽  
pp. 88-91 ◽  
Author(s):  
Kipp Weiskopf ◽  
Aaron M. Ring ◽  
Chia Chi M. Ho ◽  
Jens-Peter Volkmer ◽  
Aron M. Levin ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Immune Responses ◽  
Cancer Cells ◽  
Macrophage Activation ◽  
Universal Method ◽  
Wild Type ◽  
High Affinity ◽  
Macrophage Phagocytosis

CD47 is an antiphagocytic signal that cancer cells employ to inhibit macrophage-mediated destruction. Here, we modified the binding domain of human SIRPα, the receptor for CD47, for use as a CD47 antagonist. We engineered high-affinity SIRPα variants with about a 50,000-fold increased affinity for human CD47 relative to wild-type SIRPα. As high-affinity SIRPα monomers, they potently antagonized CD47 on cancer cells but did not induce macrophage phagocytosis on their own. Instead, they exhibited remarkable synergy with all tumor-specific monoclonal antibodies tested by increasing phagocytosis in vitro and enhancing antitumor responses in vivo. This “one-two punch” directs immune responses against tumor cells while lowering the threshold for macrophage activation, thereby providing a universal method for augmenting the efficacy of therapeutic anticancer antibodies.

scholarly journals DDRE-20. GENERATION OF A HIGH-AFFINITY EGFR/EGFRvIII-TARGETED D2C7 CAR-T CELL

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii65-ii66
Author(s):  
Katherine Ryan ◽  
Daniel Wilkinson ◽  
Daniel Landi ◽  
Peter Fecci
Keyword(s):  
T Cells ◽  
T Cell ◽  
Murine Models ◽  
Wild Type ◽  
High Affinity ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Abstract BACKGROUND The intent of this investigation is to validate a novel, high-affinity epidermal growth factor receptor (EGFR) chimeric antigen receptor (CAR) T-cell product for adoptive immunotherapy of EGFR-expressing malignancies of the central nervous system (CNS). Wild type EGFR is ubiquitously expressed on glial tumors in both children and adults, and the majority of solid tumors that metastasize to the CNS but is not expressed on healthy CNS tissues. EGFR and its isoform mutant, EGFRvIII, are hyperactivated or overexpressed receptor tyrosine kinases described in many human cancers. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) that binds to both wild type EGFR and EGFRvIII. We hypothesize that D2C7 adapted as a CAR on primary human T cells will generate potent, cytotoxic activity against EGFR-expressing tumors in vivo. METHODS In vitro interferon gamma release assays comparing the D2C7 CAR to the EGFRvIII CAR have proven that D2C7 CAR-T cells have high specificity and potent cytotoxicity against established murine glioblastoma (GBM) tumor cell lines, expressing both EGFR and EGFRvIII. Next steps include pre-clinical in vivo testing of the D2C7 CAR-T cells in murine models of CNS tumors expressing EGFR/EGFRvIII such as GBM, non-small cell lung carcinoma (NSCLC), breast carcinoma, and melanoma. CONCLUSIONS To improve and validate the effectiveness of CAR T-cell therapy for EGFR-expressing CNS tumors, our team has designed a high-affinity CAR-T cell that targets both EGFRvIII and wild-type EGFR, known as a D2C7 CAR-T cell. This investigation has established pre-clinical anti-tumor activity of D2C7-CAR T cells in vitro and we plan to present more mature data regarding efficacy in orthotopic murine models of GBM, NSCLC, breast carcinoma, and melanoma at the meeting this Fall.

scholarly journals Differential Requirement for PBP1a and PBP1b inIn VivoandIn VitroFitness of Vibrio cholerae

2014 ◽  
Vol 82 (5) ◽  
pp. 2115-2124 ◽  
Author(s):  
Tobias Dörr ◽  
Andrea Möll ◽  
Michael C. Chao ◽  
Felipe Cava ◽  
Hubert Lam ◽  
...  
Keyword(s):  
Stationary Phase ◽  
Vibrio Cholerae ◽  
Synthetic Lethality ◽  
Wild Type ◽  
Content Type ◽  
Infant Mouse ◽  
Mouse Small Intestine ◽  
Membrane Lipoproteins

ABSTRACTWe investigated the roles of theVibrio choleraehigh-molecular-weight bifunctional penicillin binding proteins, PBP1a and PBP1b, in the fitness of this enteric pathogen. Using a screen for synthetic lethality, we found that theV. choleraePBP1a and PBP1b proteins, like theirEscherichia colihomologues, are each essential in the absence of the other and in the absence of the other's putative activator, the outer membrane lipoproteins LpoA and LpoB, respectively. Comparative analyses ofV. choleraemutants suggest that PBP1a/LpoA ofV. choleraeplay a more prominent role in generating and/or maintaining the pathogen's cell wall than PBP1b/LpoB.V. choleraelacking PBP1b or LpoB exhibited wild-type growth under all conditions tested. In contrast,V. choleraelacking PBP1a or LpoA exhibited growth deficiencies in minimal medium, in the presence of deoxycholate and bile, and in competition assays with wild-type cells bothin vitroand in the infant mouse small intestine. PBP1a pathway mutants are particularly impaired in stationary phase, which renders them sensitive to a product(s) present in supernatants from stationary-phase wild-type cells. The marked competitive defect of the PBP1a pathway mutantsin vivowas largely absent when exponential-phase cells rather than stationary-phase cells were used to inoculate suckling mice. Thus, at least forV. choleraePBP1a pathway mutants, the growth phase of the inoculum is a key modulator of infectivity.

scholarly journals Direct Regulation of Bacillus subtilis phoPR Transcription by Transition State Regulator ScoC

2010 ◽  
Vol 192 (12) ◽  
pp. 3103-3113 ◽  
Author(s):  
Bindiya Kaushal ◽  
Salbi Paul ◽  
F. Marion Hulett
Keyword(s):  
Bacillus Subtilis ◽  
Transition State ◽  
Stationary Phase ◽  
Promoter Region ◽  
Regulatory Protein ◽  
Cellular Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Promoter Fusion

ABSTRACT Induction of the Pho response in Bacillus subtilis occurs when the Pi concentrations in the growth medium fall below 0.1 mM, a condition which results in slowed cellular growth followed by entry into stationary phase. The phoPR promoter region contains three σA-responsive promoters; only promoter PA4 is PhoP autoregulated. Expression of the phoPR operon is postexponential, suggesting the possibility of a repressor role for a transition-state-regulatory protein(s). Expression of a phoPR promoter-lacZ fusion in a scoC loss-of-function mutant strain grown in low-phosphate defined medium was significantly higher than expression in the wild-type strain during exponential growth or stationary phase. Derepression in the scoC strain from a phoP promoter fusion containing a mutation in the CcpA binding site (cre1) was further elevated approximately 1.4-fold, indicating that the repressor effects of ScoC and CcpA on phoP expression were cumulative. DNase I footprinting showed protection of putative binding sites by ScoC, which included the −10 and/or −35 elements of five (PB1, PE2, PA3, PA4, and PA6) of the six promoters within the phoPR promoter region. PA6 was expressed in vivo from the phoP cre1 promoter fusion in both wild-type and scoC strains. Evidence for ScoC repression in vivo was shown by primer extension for PA4 and PA3 from the wild-type promoter and for PA4 and PE2 from the phoP cre1 promoter. The latter may reflect ScoC repression of sporulation that indirectly affects phoPR transcription. ScoC was shown to repress PA6, PA4, PE2, and PB1 in vitro.

Sign in / Sign up

Export Citation Format

Share Document