(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes

ABSTRACT The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenes. IMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes. Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

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From (p)ppGpp to (pp)pGpp: Characterization of Regulatory Effects of pGpp Synthesized by the Small Alarmone Synthetase of Enterococcus faecalis

Journal of Bacteriology ◽

10.1128/jb.00324-15 ◽

2015 ◽

Vol 197 (18) ◽

pp. 2908-2919 ◽

Cited By ~ 58

Author(s):

Anthony O. Gaca ◽

Pavel Kudrin ◽

Cristina Colomer-Winter ◽

Jelena Beljantseva ◽

Kuanqing Liu ◽

...

Keyword(s):

Stress Tolerance ◽

Enterococcus Faecalis ◽

Second Messengers ◽

Stringent Response ◽

Synthetase Activity ◽

Protective Effects ◽

Content Type ◽

Regulatory Effects ◽

Complex Target

ABSTRACTThe bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. InEnterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolaseE. faecalisRel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation inFirmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEfsynthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEfalso efficiently utilized GMP to form GMP 3′-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity ofE. faecalisenzymes involved in GTP biosynthesis and, to a lesser extent, transcription ofrrnBbyEscherichia coliRNA polymerase. Activation ofE. coliRelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEfwas activated only by ppGpp. Furthermore, enzymatic activity of RelQEfis insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of “long” RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.IMPORTANCEAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ ofEnterococcus faecalis(RelQEf), we found that, in addition to (p)ppGpp, RelQEfis an efficient producer of pGpp (GMP 3′-diphosphate).In vitroanalysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEfand suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

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Hofbauer Cells Spread Listeria monocytogenes among Placental Cells and Undergo Pro-Inflammatory Reprogramming while Retaining Production of Tolerogenic Factors

mBio ◽

10.1128/mbio.01849-21 ◽

2021 ◽

Author(s):

Siavash Azari ◽

Lauren J. Johnson ◽

Amy Webb ◽

Sophia M. Kozlowski ◽

Xiaoli Zhang ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Pregnancy Complications ◽

Intracellular Pathogen ◽

Content Type ◽

Placental Cells ◽

Hofbauer Cells ◽

Anti Inflammatory

Infection of the placental/fetal unit by the facultative intracellular pathogen Listeria monocytogenes results in severe pregnancy complications. Hofbauer cells (HBCs) are fetal macrophages that play homeostatic anti-inflammatory functions in healthy placentas.

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Investigating the Roles of Listeria monocytogenes Peroxidases in Growth and Virulence

Microbiology Spectrum ◽

10.1128/spectrum.00440-21 ◽

2021 ◽

Author(s):

Monica R. Cesinger ◽

Nicole H. Schwardt ◽

Cortney R. Halsey ◽

Maureen K. Thomason ◽

Michelle L. Reniere

Keyword(s):

Reactive Oxygen Species ◽

Immune System ◽

Listeria Monocytogenes ◽

Causative Agent ◽

Foodborne Illness ◽

Intracellular Pathogen ◽

Host Immune System ◽

Oxygen Species ◽

Aerobic Growth ◽

Content Type

Listeria monocytogenes is a facultative intracellular pathogen and the causative agent of the foodborne illness listeriosis. L. monocytogenes must contend with reactive oxygen species generated extracellularly during aerobic growth and intracellularly by the host immune system. However, the mechanisms by which L. monocytogenes defends against peroxide toxicity have not yet been defined.

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Improvement of Immunogenicity of Meningococcal Lipooligosaccharide by Coformulation with Lipidated Transferrin-Binding Protein B in Liposomes: Implications for Vaccine Development

Clinical and Vaccine Immunology ◽

10.1128/cvi.05683-11 ◽

2012 ◽

Vol 19 (5) ◽

pp. 711-722 ◽

Cited By ~ 3

Author(s):

Noëlle Mistretta ◽

Bruno Guy ◽

Yves Bérard ◽

François Dalençon ◽

Olivia Fratantonio ◽

...

Keyword(s):

Vaccine Development ◽

Binding Protein ◽

Hek293 Cells ◽

Molar Ratio ◽

Adjuvant Effect ◽

Content Type ◽

Endotoxic Activity ◽

Protein B

ABSTRACTAmong various meningococcal antigens, lipooligosaccharide (LOS) and recombinant lipidated transferrin-binding protein B (rlip-TbpB) are considered to be putative vaccine candidates against group BNeisseria meningitidis. In the present work, we report the development of a new liposome-based vaccine formulation containing both rlip-TbpB and L8 LOS. The endotoxic activity of the liposomal LOS was evaluatedin vitrousing theLimulusAmebocyte Lysate assay and compared to the endotoxic activity of free LOS. Above a 250:1 lipid/LOS molar ratio, liposomes were shown to effectively detoxify the LOS as the endotoxic activity of the LOS was reduced by more than 99%. Immunogenicity studies in rabbits showed that the presence of rlip-TbpB dramatically increased the immunogenicity of the LOS. While the formulation raised a strong anti-TbpB response, it elicited a higher anti-LOS IgG level than the liposomal LOS alone. Sera from rabbits immunized with rlip-TbpB/liposomal LOS displayed increased ability to recognize LOS on live bacteria expressing the L8 immunotype and increased anti-LOS-specific bactericidal activity compared to sera from rabbits immunized with liposomal LOS alone. Measurement of interleukin-8 (IL-8) produced by HEK293 cells transfected with Toll-like receptor (TLR) after stimulation with rlip-TbpB showed that the protein is a TLR2 agonist, which is in accordance with the structure of its lipid. Furthermore, anin vivostudy demonstrated that the lipid moiety is not only required for its adjuvant effect but also has to be linked to the protein. Overall, the rlip-TbpB/LOS liposomal formulation was demonstrated to induce an effective anti-LOS response due to the adjuvant effect of rlip-TbpB on LOS.

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Transferrin Binding Protein B and Transferrin Binding Protein A2 Expand the Transferrin Recognition Range of Histophilus somni

Journal of Bacteriology ◽

10.1128/jb.00177-20 ◽

2020 ◽

Vol 202 (14) ◽

Author(s):

Anastassia K. Pogoutse ◽

Trevor F. Moraes

Keyword(s):

Host Species ◽

Binding Protein ◽

Iron Acquisition ◽

Acquisition System ◽

Content Type ◽

Sheep And Goats ◽

Iron Acquisition System ◽

Histophilus Somni ◽

Bovine Transferrin ◽

Protein B

ABSTRACT The bacterial bipartite transferrin receptor is an iron acquisition system that several important human and animal pathogens require for survival. It consists of the TonB-dependent transporter transferrin binding protein A (TbpA) and the surface lipoprotein transferrin binding protein B (TbpB). Curiously, the Tbps are only found in host-specific pathogens and are themselves host specific, meaning that they will bind to the transferrin of their host species but not to the transferrins of other animal species. While this phenomenon has long been established, neither the steps in the evolutionary process that led to this exquisite adaptation for the host nor the steps that could alter it are known. We sought to gain insight into these processes by studying Tbp specificity in Histophilus somni, an economically important pathogen of cattle. A past study showed that whole cells of H. somni specifically bind bovine transferrin but not transferrin from sheep and goats, two bovids whose transferrins share 93% amino acid sequence identity with bovine transferrin. To our surprise, we found that H. somni can use sheep and goat transferrins as iron sources for growth and that HsTbpB, but not HsTbpA, has detectable affinity for sheep and goat transferrins. Furthermore, a third transferrin binding protein found in H. somni, HsTbpA2, also showed affinity for sheep and goat transferrins. Our results suggest that H. somni TbpB and TbpA2 may contribute to broadening the host transferrin recognition range of H. somni. IMPORTANCE Host-restricted pathogens infect a single host species or a narrow range of host species. Histophilus somni, a pathogen that incurs severe economic losses for the cattle industry, infects cattle, sheep, and goats but not other mammals. The transferrin binding proteins, TbpA and TbpB, are thought to be a key iron acquisition system in H. somni; however, despite their importance, H. somni TbpA and TbpB were previously shown to be cattle transferrin specific. In our study, we find that H. somni TbpB and another little-studied Tbp, TbpA2, bind sheep and goat transferrins, as well as bovine transferrin. Our results suggest that TbpB and TbpA2 may allow for host range expansion and provide a mechanism for how host specificity in Tbp-encoding pathogens can be altered.

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Nonbinding Site-Directed Mutants of Transferrin Binding Protein B Exhibit Enhanced Immunogenicity and Protective Capabilities

Infection and Immunity ◽

10.1128/iai.02572-14 ◽

2014 ◽

Vol 83 (3) ◽

pp. 1030-1038 ◽

Cited By ~ 31

Author(s):

Rafael Frandoloso ◽

Sonia Martínez-Martínez ◽

Charles Calmettes ◽

Jamie Fegan ◽

Estela Costa ◽

...

Keyword(s):

Immune Response ◽

Binding Protein ◽

Iron Acquisition ◽

Critical Role ◽

T Cell Response ◽

Content Type ◽

Growth And Survival ◽

Mutant Proteins ◽

Protein B

Host-adapted Gram-negative bacterial pathogens from thePasteurellaceae,Neisseriaceae, andMoraxellaceaefamilies normally reside in the upper respiratory or genitourinary tracts of their hosts and rely on utilizing iron from host transferrin (Tf) for growth and survival. The surface receptor proteins that mediate this critical iron acquisition pathway have been proposed as ideal vaccine targets due to the critical role that they play in survival and disease pathogenesisin vivo. In particular, the surface lipoprotein component of the receptor, Tf binding protein B (TbpB), had received considerable attention as a potential antigen for vaccines in humans and food production animals but this has not translated into the series of successful vaccine products originally envisioned. Preliminary immunization experiments suggesting that host Tf could interfere with development of the immune response prompted us to directly address this question with site-directed mutant proteins defective in binding Tf. Site-directed mutants with dramatically reduced binding of porcine transferrin and nearly identical structure to the native proteins were prepared. A mutantHaemophilus parasuisTbpB was shown to induce an enhanced B-cell and T-cell response in pigs relative to native TbpB and provide superior protection from infection than the native TbpB or a commercial vaccine product. The results indicate that binding of host transferrin modulates the development of the immune response against TbpBs and that strategies designed to reduce or eliminate binding can be used to generate superior antigens for vaccines.

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The secRNome of Listeria monocytogenes Harbors Small Noncoding RNAs That Are Potent Inducers of Beta Interferon

mBio ◽

10.1128/mbio.01223-19 ◽

2019 ◽

Vol 10 (5) ◽

Cited By ~ 12

Author(s):

Renate Frantz ◽

Lisa Teubner ◽

Tilman Schultze ◽

Luigi La Pietra ◽

Christin Müller ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Bacterial Growth ◽

Cell Envelope ◽

Intracellular Pathogen ◽

Type I ◽

Type I Ifn ◽

Intracellular Growth ◽

Potent Inducer ◽

Content Type ◽

Bacterial Physiology

ABSTRACT Cellular sensing of bacterial RNA is increasingly recognized as a determinant of host-pathogen interactions. The intracellular pathogen Listeria monocytogenes induces high levels of type I interferons (alpha/beta interferons [IFN-α/β]) to create a growth-permissive microenvironment during infection. We previously demonstrated that RNAs secreted by L. monocytogenes (comprising the secRNome) are potent inducers of IFN-β. We determined the composition and diversity of the members of the secRNome and found that they are uniquely enriched for noncoding small RNAs (sRNAs). Testing of individual sRNAs for their ability to induce IFN revealed several sRNAs with this property. We examined ril32, an intracellularly expressed sRNA that is highly conserved for the species L. monocytogenes and that was the most potent inducer of IFN-β expression of all the sRNAs tested in this study, in more detail. The rli32-induced IFN-β response is RIG-I (retinoic acid inducible gene I) dependent, and cells primed with rli32 inhibit influenza virus replication. We determined the rli32 motif required for IFN induction. rli32 overproduction promotes intracellular bacterial growth, and a mutant lacking rli32 is restricted for intracellular growth in macrophages. rli32-overproducing bacteria are resistant to H2O2 and exhibit both increased catalase activity and changes in the cell envelope. Comparative transcriptome sequencing (RNA-Seq) analysis indicated that ril32 regulates expression of the lhrC locus, previously shown to be involved in cell envelope stress. Inhibition of IFN-β signaling by ruxolitinib reduced rli32-dependent intracellular bacterial growth, indicating a link between induction of the interferon system and bacterial physiology. rli32 is, to the best of our knowledge, the first secreted individual bacterial sRNA known to trigger the induction of the type I IFN response. IMPORTANCE Interferons are potent and broadly acting cytokines that stimulate cellular responses to nucleic acids of unusual structures or locations. While protective when induced following viral infections, the induction of interferons is detrimental to the host during L. monocytogenes infection. Here, we identify specific sRNAs, secreted by the bacterium, with the capacity to induce type I IFN. Further analysis of the most potent sRNA, rli32, links the ability to induce RIG-I-dependent induction of the type I IFN response to the intracellular growth properties of the bacterium. Our findings emphasize the significance of released RNA for Listeria infection and shed light on a compartmental strategy used by an intracellular pathogen to modulate host responses to its advantage.

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Intracellular Listeria monocytogenes Comprises a Minimal but Vital Fraction of the Intestinal Burden following Foodborne Infection

Infection and Immunity ◽

10.1128/iai.00503-15 ◽

2015 ◽

Vol 83 (8) ◽

pp. 3146-3156 ◽

Cited By ~ 14

Author(s):

Grant S. Jones ◽

Kate M. Bussell ◽

Tanya Myers-Morales ◽

Abigail M. Fieldhouse ◽

Elsa N. Bou Ghanem ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Wild Type Strain ◽

Intracellular Pathogen ◽

Mesenteric Lymph Nodes ◽

Free Living ◽

Content Type ◽

Foodborne Infection ◽

Mammalian Infection ◽

Foodborne Infections

Listeria monocytogenesis a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle ofL. monocytogenesis considered to be its primary virulence determinant during mammalian infection; however, the proportion ofL. monocytogenesthat is intracellularin vivohas not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlAm-expressing)L. monocytogenesrecovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, significantly lower burdens ofL. monocytogeneswere recovered from the colon, spleen, and liver of gentamicin-treated mice than of control mice. This led us to investigate whether intracellular replication ofL. monocytogeneswas essential during the intestinal phase of infection. We found that lipoate protein ligase-deficientL. monocytogenes(ΔlplA1) mutants, which display impaired intracellular growth, were able to colonize the colon but did not persist efficiently and had a significant defect in spreading to the MLN, spleen, and liver. Together, these data indicate that the majority of theL. monocytogenesburden in the gastrointestinal tract is extracellular, but the small proportion of intracellularL. monocytogenesis essential for dissemination to the MLN and systemic organs.

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Expression of Surface Protein LapB by a Wide Spectrum of Listeria monocytogenes Serotypes as Demonstrated with Anti-LapB Monoclonal Antibodies

Applied and Environmental Microbiology ◽

10.1128/aem.01908-16 ◽

2016 ◽

Vol 82 (22) ◽

pp. 6768-6778 ◽

Cited By ~ 2

Author(s):

Teela Boivin ◽

Cathie Elmgren ◽

Brian W. Brooks ◽

Hongsheng Huang ◽

Franco Pagotto ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Listeria Monocytogenes ◽

Bioinformatics Analysis ◽

Polyclonal Antibodies ◽

Surface Protein ◽

Surface Proteins ◽

Content Type ◽

Adhesion Protein ◽

Gene Coding ◽

Protein B

ABSTRACTProtein antigens expressed on the surface of all strains ofListeria monocytogenesand absent from nonpathogenicListeriaspp. are presumably useful targets for pathogen identification, detection, and isolation using specific antibodies (Abs). To seek such surface proteins expressed in various strains ofL. monocytogenesfor diagnostic applications, we focused on a set of surface proteins known to be involved or putatively involved inL. monocytogenesvirulence and identifiedListeriaadhesion protein B (LapB) as a candidate based on the bioinformatics analysis of whole-genome sequences showing that the gene coding for LapB was present inL. monocytogenesstrains and absent from strains of otherListeriaspp. Immunofluorescence microscopy (IFM), performed with rabbit polyclonal antibodies against the recombinant LapB protein (rLapB) ofL. monocytogenesserotype 4b strain L10521, confirmed expression of LapB on the surface. A panel of 48 mouse monoclonal antibodies (MAbs) to rLaB was generated, and 7 of them bound strongly to the surface ofL. monocytogenescells as demonstrated using IFM. Further characterization of these 7 anti-LapB MAbs, using an enzyme-linked immunosorbent assay (ELISA), revealed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) reacted strongly with 46 (86.8%) of 53 strains representing 10 of the 12 serotypes tested (1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4ab, 4b, 4d, and 4e). These results indicate that LapB, together with companion anti-LapB MAbs, can be targeted as a biomarker for the detection and isolation of variousL. monocytogenesstrains from contaminated foods.IMPORTANCEStrains ofL. monocytogenesare traditionally grouped into serotypes. Identification of a surface protein expressed in all or the majority of at least 12 serotypes would aid in the development of surface-binding monoclonal antibodies (MAbs) for detection and isolation ofL. monocytogenesfrom foods. Bioinformatics analysis revealed that the gene coding forListeriaadhesion protein B (LapB), a surface protein involved inL. monocytogenesvirulence, was present inL. monocytogenesstrains and absent from otherListeriaspp. Polyclonal antibodies against recombinant LapB (rLapB) detected the exposed epitopes on the surface ofL. monocytogenes. Production and extensive assessment of 48 MAbs to rLapB showed that 6 anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, and M3519) detected the expression of LapB in a wide range ofL. monocytogenesisolates representing 10 of 12 serotypes tested, suggesting that LapB, together with specific MAbs, can be targeted as a biomarker for pathogen detection and isolation.

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RelA Inhibits Bacillus subtilis Motility and Chaining

Journal of Bacteriology ◽

10.1128/jb.02063-14 ◽

2014 ◽

Vol 197 (1) ◽

pp. 128-137 ◽

Cited By ~ 17

Author(s):

Qutaiba O. Ababneh ◽

Jennifer K. Herman

Keyword(s):

Bacillus Subtilis ◽

Second Messengers ◽

Cell Formation ◽

Stringent Response ◽

Alternative Sigma Factor ◽

Content Type ◽

Biofilm Production ◽

Motile Cells ◽

Persister Cell ◽

Developmental Decision

The nucleotide second messengers pppGpp and ppGpp [(p)ppGpp] are responsible for the global downregulation of transcription, translation, DNA replication, and growth rate that occurs during the stringent response. More recent studies suggest that (p)ppGpp is also an important effector in many nonstringent processes, including virulence, persister cell formation, and biofilm production. InBacillus subtilis, (p)ppGpp production is primarily determined by the net activity of RelA, a bifunctional (p)ppGpp synthetase/hydrolase, and two monofunctional (p)ppGpp synthetases, YwaC and YjbM. We observe that inB. subtilis, arelAmutant grows exclusively as unchained, motile cells, phenotypes regulated by the alternative sigma factor SigD. Our data indicate that therelAmutant is trapped in a SigD “on” state during exponential growth, implicating RelA and (p)ppGpp levels in the regulation of cell chaining and motility inB. subtilis. Our results also suggest that minor variations in basal (p)ppGpp levels can significantly skew developmental decision-making outcomes.

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