Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

ABSTRACTCarbon storage regulator A ofBorrelia burgdorferi(CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABbare dependent on environmental signals and on select residues. We analyzed the phenotype ofcsrABbdeletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABbin thecsrABbmutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in thecsrABbmutant to parental levels, indicating that both the levels of CsrABband the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb(8S) with alanines resulted in increased levels of CsrABband reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members ofrpoSregulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb(7D) resulted in reducedcsrABbtranscripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream ofptacompared to the parental and 7D truncated protein. Two CsrABbbinding sites were also identified upstream offliWwithin theflgKcoding sequence. These observations reveal conserved and unique functions of CsrABbthat regulate adaptive gene expression inB. burgdorferi.

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Inactivation ofbb0184, Which Encodes Carbon Storage Regulator A, Represses the Infectivity ofBorrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.00871-10 ◽

2010 ◽

Vol 79 (3) ◽

pp. 1270-1279 ◽

Cited By ~ 42

Author(s):

Ching Wooen Sze ◽

Chunhao Li

Keyword(s):

Carbon Storage ◽

Associated Factors ◽

Animal Studies ◽

Acetate Metabolism ◽

Acetyl Phosphate ◽

Wild Type ◽

Acetyl Coa ◽

Acetyl Coenzyme A ◽

Carbon Storage Regulator ◽

Subsequent Activation

ABSTRACTThe genome ofBorrelia burgdorferi, the Lyme disease spirochete, encodes a homolog (thebb0184gene product) of the carbon storage regulator A protein (CsrABb); recent studies reported that CsrABbis involved in the regulation of several infectivity factors ofB. burgdorferi. However, the mechanism involved remains unknown. In this report, acsrABbmutant was constructed and complemented in an infectious B31A3 strain. Subsequent animal studies showed that the mutant failed to establish an infection in mice, highlighting that CsrABbis required for the infectivity ofB. burgdorferi. Western blot analyses revealed that the virulence-associated factors OspC, DbpB, and DbpA were attenuated in thecsrABbmutant. The Rrp2-RpoN-RpoS pathway (σ54-σSsigma factor cascade) is a central regulon that governs the expression ofospC,dbpB, anddbpA. Further analyses found that the level of RpoS was significantly decreased in the mutant, while the level of Rrp2 remained unchanged. A recent study reported that the overexpression of BB0589, a phosphate acetyl-transferase (Pta) that converts acetyl-phosphate to acetyl-coenzyme A (CoA), led to the inhibition of RpoS and OspC expression, suggesting that acetyl-phosphate is an activator of Rrp2. Along with this report, we found that CsrABbbinds to the leader sequence of thebb0589transcript and that the intracellular level of acetyl-CoA in thecsrABbmutant was significantly increased compared to that of the wild type, suggesting that more acetyl-phosphate was being converted to acetyl-CoA in the mutant. Collectively, these results suggest that CsrABbmay influence the infectivity ofB. burgdorferivia regulation of acetate metabolism and subsequent activation of the Rrp2-RpoN-RpoS pathway.

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CsrA (BB0184) Is Not Involved in Activation of the RpoN-RpoS Regulatory Pathway in Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.01555-13 ◽

2014 ◽

Vol 82 (4) ◽

pp. 1511-1522 ◽

Cited By ~ 10

Author(s):

Zhiming Ouyang ◽

Jianli Zhou ◽

Michael V. Norgard

Keyword(s):

Gene Expression ◽

Borrelia Burgdorferi ◽

Animal Studies ◽

Expression System ◽

Regulatory Pathway ◽

Content Type ◽

Mouse Tissues ◽

Differential Gene ◽

Carbon Storage Regulator

ABSTRACTBorrelia burgdorferiencodes a homologue of the bacterial carbon storage regulator A (CsrA). Recently, it was reported that CsrA contributes toB. burgdorferiinfectivity and is required for the activation of the central RpoN-RpoS regulatory pathway. However, many questions concerning the function of CsrA inB. burgdorferigene regulation remain unanswered. In particular, there are conflicting reports concerning the molecular details of how CsrA may modulaterpoSexpression and, thus, how CsrA may influence the RpoN-RpoS pathway inB. burgdorferi. To address these key discrepancies, we examined the role of CsrA in differential gene expression in the Lyme disease spirochete. Upon engineering an induciblecsrAexpression system inB. burgdorferi, controlled hyperexpression of CsrA in a merodiploid strain did not significantly alter the protein and transcript levels ofbosR,rpoS, and RpoS-dependent genes (such asospCanddbpA). In addition, we constructed isogeniccsrAmutants in two widely used infectiousB. burgdorferistrains. When expression ofbosR,rpoS,ospC, anddbpAwas compared between thecsrAmutants and their wild-type counterparts, no detectable differences were observed. Finally, animal studies indicated that thecsrAmutants remained infectious for and virulent in mice. Analyses ofB. burgdorferigene expression in mouse tissues showed comparable levels ofrpoStranscripts by thecsrAmutants and the parental strains. Taken together, these results constitute compelling evidence that CsrA is not involved in activation of the RpoN-RpoS pathway and is dispensable for mammalian infectious processes carried out byB. burgdorferi.

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An Ixodes scapularis Protein Disulfide Isomerase Contributes to Borrelia burgdorferi Colonization of the Vector

Infection and Immunity ◽

10.1128/iai.00426-20 ◽

2020 ◽

Vol 88 (12) ◽

Author(s):

Yongguo Cao ◽

Connor Rosen ◽

Gunjan Arora ◽

Akash Gupta ◽

Carmen J. Booth ◽

...

Keyword(s):

Life Cycle ◽

Borrelia Burgdorferi ◽

Protein Disulfide Isomerase ◽

Inflammatory Responses ◽

Ixodes Scapularis ◽

Vertebrate Host ◽

Content Type ◽

Disulfide Isomerase ◽

Complex Interactions ◽

Protein Disulfide

ABSTRACT Borrelia burgdorferi causes Lyme disease, the most common tick-transmitted illness in North America. When Ixodes scapularis feed on an infected vertebrate host, spirochetes enter the tick gut along with the bloodmeal and colonize the vector. Here, we show that a secreted tick protein, I. scapularis protein disulfide isomerase A3 (IsPDIA3), enhances B. burgdorferi colonization of the tick gut. I. scapularis ticks in which ispdiA3 has been knocked down using RNA interference have decreased spirochete colonization of the tick gut after engorging on B. burgdorferi-infected mice. Moreover, administration of IsPDIA3 antiserum to B. burgdorferi-infected mice reduced the ability of spirochetes to colonize the tick when feeding on these animals. We show that IsPDIA3 modulates inflammatory responses at the tick bite site, potentially facilitating spirochete survival at the vector-host interface as it exits the vertebrate host to enter the tick gut. These data provide functional insights into the complex interactions between B. burgdorferi and its arthropod vector and suggest additional targets to interfere with the spirochete life cycle.

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Carbon storage regulator A (CsrABb) is a repressor of Borrelia burgdorferi flagellin protein FlaB

Molecular Microbiology ◽

10.1111/j.1365-2958.2011.07853.x ◽

2011 ◽

Vol 82 (4) ◽

pp. 851-864 ◽

Cited By ~ 41

Author(s):

Ching Wooen Sze ◽

Dustin R. Morado ◽

Jun Liu ◽

Nyles W. Charon ◽

Hongbin Xu ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Carbon Storage ◽

Carbon Storage Regulator

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Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms

Infection and Immunity ◽

10.1128/iai.01239-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 608-617 ◽

Cited By ~ 13

Author(s):

Dharanesh Gangaiah ◽

Wei Li ◽

Kate R. Fortney ◽

Diane M. Janowicz ◽

Sheila Ellinger ◽

...

Keyword(s):

Oxidative Stress ◽

Stationary Phase ◽

Carbon Storage ◽

Stress Responses ◽

Haemophilus Ducreyi ◽

Content Type ◽

Enhanced Sensitivity ◽

Significant Survival ◽

Carbon Storage Regulator ◽

Mutant Phenotypes

ABSTRACTThe carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence.Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog ofcsrA. Here, we generated an unmarked, in-frame deletion mutant ofcsrAto assess its contribution toH. ducreyipathogenesis. In human inoculation experiments, thecsrAmutant was partially attenuated for pustule formation compared to its parent. Deletion ofcsrAresulted in decreased adherence ofH. ducreyito human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants ofH. ducreyiadherence to HFF cells, were downregulated in thecsrAmutant. Compared to its parent, thecsrAmutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. ThecsrAmutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation intranspartially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

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Oligopeptide Permease A5 Modulates Vertebrate Host-Specific Adaptation of Borrelia burgdorferi

Infection and Immunity ◽

10.1128/iai.05234-11 ◽

2011 ◽

Vol 79 (8) ◽

pp. 3407-3420 ◽

Cited By ~ 23

Author(s):

B. V. Subba Raju ◽

Maria D. Esteve-Gassent ◽

S. L. Rajasekhar Karna ◽

Christine L. Miller ◽

Tricia A. Van Laar ◽

...

Keyword(s):

Borrelia Burgdorferi ◽

Shuttle Vector ◽

Immunoblot Analysis ◽

Vertebrate Host ◽

Transcriptional Analysis ◽

Wild Type ◽

Content Type ◽

Specific Adaptation ◽

Protein Levels ◽

Oligopeptide Permease

ABSTRACTBorrelia burgdorferi, the agent of Lyme disease, undergoes rapid adaptive gene expression in response to signals unique to its arthropod vector or vertebrate hosts. Among the upregulated genes under vertebrate host conditions is one of the five annotated homologs of oligopeptide permease A (OppA5, BBA34). A mutant lackingoppA5was constructed in an lp25-deficient isolate ofB. burgdorferistrain B31, and the minimal regions of infectivity were restored via a shuttle vector pBBE22 with or without an intact copy ofbba34. Immunoblot analysis of thebba34mutant revealed a reduction in the levels of RpoS, BosR, and CsrABbwith a concomitant reduction in the levels of OspC, DbpA, BBK32, and BBA64. There were no changes in the levels of OspA, NapA, P66, and three other OppA orthologs. Quantitative transcriptional analysis correlated with the changes in the protein levels. However, thebba34mutant displayed comparable infectivities in the C3H/HeN mice and the wild-type strain, despite the reduction in several pathogenesis-related proteins. Supplementation of the growth medium with increased levels of select components, notably sodium acetate and sodium bicarbonate, restored the levels of several proteins in thebba34mutant to wild-type levels. We speculate that the transport of acetate appears to contribute to the accumulation of key metabolites, like acetyl phosphate, that facilitate the adaptation ofB. burgdorferito the vertebrate host by the activation of the Rrp2-RpoN-RpoS pathway. These studies underscore the importance of solute transport to host-specific adaptation ofB. burgdorferi.

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A MarR-Type Regulator Directly Activates Transcription from the Brucella abortus virB Promoter by Sharing a Redundant Role with HutC

Journal of Bacteriology ◽

10.1128/jb.01007-12 ◽

2012 ◽

Vol 194 (23) ◽

pp. 6431-6440 ◽

Cited By ~ 7

Author(s):

Rodrigo Sieira ◽

Gastón M. Arocena ◽

Angeles Zorreguieta ◽

Diego J. Comerci ◽

Rodolfo A. Ugalde

Keyword(s):

Transcription Factors ◽

Dna Sequences ◽

Cell Envelope ◽

Regulatory Element ◽

Dependent Manner ◽

Secretion Systems ◽

Mobility Shift ◽

Content Type ◽

Environmental Signals

ABSTRACTType IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genusBrucellaessentially depends on the integrity of the T4SS-encodingvirBoperon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of thevirBgenes that were isolated from total protein extracts ofBrucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of thevirBpromoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNAin vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activatedvirBexpression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of thevirBpromoter and is probably involved in coordinating gene expression in response to specific environmental signals.

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Glucose-Specific Enzyme IIA Has Unique Binding Partners in The Vibrio cholerae Biofilm

mBio ◽

10.1128/mbio.00228-12 ◽

2012 ◽

Vol 3 (6) ◽

Cited By ~ 22

Author(s):

Bradley S. Pickering ◽

Daniel R. Smith ◽

Paula I. Watnick

Keyword(s):

Adenylate Cyclase ◽

Vibrio Cholerae ◽

Carbon Storage ◽

Biofilm Formation ◽

Specific Enzyme ◽

Microbial Physiology ◽

Content Type ◽

Multiprotein Complexes ◽

Binding Partners ◽

Carbon Storage Regulator

ABSTRACTGlucose-specific enzyme IIA (EIIAGlc) is a central regulator of bacterial metabolism and an intermediate in the phosphoenolpyruvate phosphotransferase system (PTS), a conserved phosphotransfer cascade that controls carbohydrate transport. We previously reported that EIIAGlcactivates transcription of the genes required forVibrio choleraebiofilm formation. While EIIAGlcmodulates the function of many proteins through a direct interaction, none of the known regulatory binding partners of EIIAGlcactivates biofilm formation. Therefore, we used tandem affinity purification (TAP) to compare binding partners of EIIAGlcin both planktonic and biofilm cells. A surprising number of novel EIIAGlcbinding partners were identified predominantly under one condition or the other. Studies of planktonic cells revealed established partners of EIIAGlc, such as adenylate cyclase and glycerol kinase. In biofilms, MshH, a homolog ofEscherichia coliCsrD, was found to be a dominant binding partner of EIIAGlc. Further studies revealed that MshH inhibits biofilm formation. This function was independent of the Carbon storage regulator (Csr) pathway and dependent on EIIAGlc. To explore the existence of multiprotein complexes centered on EIIAGlc, we also affinity purified the binding partners of adenylate cyclase from biofilm cells. In addition to EIIAGlc, this analysis yielded many of the same proteins that copurified with EIIAGlc. We hypothesize that EIIAGlcserves as a hub for multiprotein complexes and furthermore that these complexes may provide a mechanism for competitive and cooperative interactions between binding partners.IMPORTANCEEIIAGlcis a global regulator of microbial physiology that acts through direct interactions with other proteins. This work represents the first demonstration that the protein partners of EIIAGlcare distinct in the microbial biofilm. Furthermore, it provides the first evidence that EIIAGlcmay exist in multiprotein complexes with its partners, setting the stage for an investigation of how the multiple partners of EIIAGlcinfluence one another. Last, it provides a connection between the phosphoenolpyruvate phosphotransferase (PTS) and Csr (Carbon storage regulator) regulatory systems. This work increases our understanding of the complexity of regulation by EIIAGlcand provides a link between the PTS and Csr networks, two global regulatory cascades that influence microbial physiology.

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Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum

Eukaryotic Cell ◽

10.1128/ec.00007-15 ◽

2015 ◽

Vol 14 (7) ◽

pp. 652-660

Author(s):

Tonya Taylor ◽

Cheryl Ingram-Smith ◽

Kerry S. Smith

Keyword(s):

Phytophthora Ramorum ◽

Regulatory Domain ◽

Kinetic Characterization ◽

Acetyl Phosphate ◽

Acetyl Coa ◽

Content Type ◽

Regulatory Domains ◽

Key Enzyme ◽

Acetyl Group

ABSTRACT Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and P i . Two classes of Pta have been identified based on the absence (Pta I ) or presence (Pta II ) of an N-terminal regulatory domain. Pta I has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica Pta II enzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycete Phytophthora ramorum . The two Ptas from P. ramorum , designated PrPta II 1 and PrPta II 2, both belong to class II. PrPta II 1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPta II 1 and the S. enterica Pta II and found that, although the N-terminal regulatory domains share only 19% identity, both enzymes are inhibited by ATP, NADP, NADH, phosphoenolpyruvate (PEP), and pyruvate in the acetyl-CoA/P i -forming direction but are differentially regulated by AMP. Phylogenetic analysis of bacterial, archaeal, and eukaryotic sequences identified four subtypes of Pta II based on the presence or absence of the P-loop and DRTGG subdomains within the N-terminal regulatory domain. Although the E. coli , S. enterica , and P. ramorum enzymes all belong to the IIa subclass, our kinetic analysis has indicated that enzymes within a subclass can still display differences in their allosteric regulation.

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Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp.

PLoS ONE ◽

10.1371/journal.pone.0260981 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0260981

Author(s):

Theerapat Phoka ◽

Lenka Fule ◽

Juliana Pipoli Da Fonseca ◽

Thomas Cokelaer ◽

Mathieu Picardeau ◽

...

Keyword(s):

Carbon Storage ◽

Environmental Changes ◽

Critical Role ◽

Differentially Expressed ◽

Global Regulator ◽

Mobility Shift ◽

Poor Growth ◽

Carbon Storage Regulator ◽

Overexpressing Strain

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

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