scholarly journals CTL0511 from Chlamydia trachomatis Is a Type 2C Protein Phosphatase with Broad Substrate Specificity

2016 ◽  
Vol 198 (13) ◽  
pp. 1827-1836 ◽  
Author(s):  
Ja E. Claywell ◽  
Derek J. Fisher
Keyword(s):  
Chlamydia Trachomatis ◽  
Substrate Specificity ◽  
Protein Phosphorylation ◽  
Phosphatase Activity ◽  
Protein Phosphatase ◽  
Bacterial Pathogens ◽  
Dependent Manner ◽  
Content Type ◽  
Bacterial Physiology ◽  
Broad Substrate Specificity

ABSTRACTProtein phosphorylation has become increasingly recognized for its role in regulating bacterial physiology and virulence.Chlamydiaspp. encode two validated Hanks'-type Ser/Thr protein kinases, which typically function with cognate protein phosphatases and appear capable of global protein phosphorylation. Consequently, we sought to identify a Ser/Thr protein phosphatase partner for the chlamydial kinases. CTL0511 fromChlamydia trachomatisL2 434/Bu, which has homologs in all sequencedChlamydiaspp., is a predicted type 2C Ser/Thr protein phosphatase (PP2C). Recombinant maltose-binding protein (MBP)-tagged CTL0511 (rCTL0511) hydrolyzedp-nitrophenyl phosphate (pNPP), a generic phosphatase substrate, in a MnCl2-dependent manner at physiological pH. Assays using phosphopeptide substrates revealed that rCTL0511 can dephosphorylate phosphorylated serine (P-Ser), P-Thr, and P-Tyr residues using either MnCl2or MgCl2, indicating that metal usage can alter substrate preference. Phosphatase activity was unaffected by PP1, PP2A, and PP3 phosphatase inhibitors, while mutation of conserved PP2C residues significantly inhibited activity. Finally, phosphatase activity was detected in elementary body (EB) and reticulate body (RB) lysates, supporting a role for protein dephosphorylation in chlamydial development. These findings support that CTL0511 is a metal-dependent protein phosphatase with broad substrate specificity, substantiating a reversible phosphorylation network inC. trachomatis.IMPORTANCEChlamydiaspp. are obligate intracellular bacterial pathogens responsible for a variety of diseases in humans and economically important animal species. Our work demonstrates thatChlamydiaspp. produce a PP2C capable of dephosphorylating P-Thr, P-Ser, and P-Tyr and thatChlamydia trachomatisEBs and RBs possess phosphatase activity. In conjunction with the chlamydial Hanks'-type kinases Pkn1 and PknD, validation of CTL0511 fulfills the enzymatic requirements for a reversible phosphoprotein network. As protein phosphorylation regulates important cellular processes, including metabolism, differentiation, and virulence, in other bacterial pathogens, these results set the stage for elucidating the role of global protein phosphorylation in chlamydial physiology and virulence.

scholarly journals Inhibition of the Protein Phosphatase CppA Alters Development ofChlamydia trachomatis

2018 ◽  
Vol 200 (19) ◽  
Author(s):  
Ja E. Claywell ◽  
Lea M. Matschke ◽  
Kyle N. Plunkett ◽  
Derek J. Fisher
Keyword(s):  
Protein Phosphorylation ◽  
Protein Phosphatase ◽  
Pathogenic Bacteria ◽  
Infection Model ◽  
Ocular Infection ◽  
Developmental Cycle ◽  
Missense Mutations ◽  
Content Type ◽  
Bacterial Physiology

ABSTRACTChlamydiae are obligate intracellular Gram-negative bacterial pathogens that undergo an essential, but poorly understood, biphasic developmental cycle transitioning between the infectious elementary body and the replicative reticulate body. Ser/Thr/Tyr phosphorylation has been increasingly recognized for its role in regulating bacterial physiology.Chlamydiaspp. encode two Hanks'-type kinases in addition to a type 2C protein phosphatase (PP2C; CppA) and appears capable of global protein phosphorylation. While these findings substantiate the importance of protein phosphorylation inChlamydia, the physiological impact of protein phosphorylation remains enigmatic. In this study, we investigated thein vivorole of CppA by using recombinant protein point mutants and small-molecule inhibitors. Recombinant CppA (rCppA) amino acid point mutants based upon missense mutations identified in growth-deficientChlamydia trachomatisstrains exhibited reduced, but not a complete loss of, phosphatase activity towardp-nitrophenyl phosphate (pNPP) and phosphopeptides. To more directly explore the importance of CppA in chlamydial development, we implemented a chemical “knockout” approach using derivatives of 5,5′-methylenedisalicylic acid (MDSA). Several MDSA derivatives significantly reduced CppA activityin vitroand the growth ofC. trachomatisL2,C. trachomatisD, andChlamydia muridarumin a cell culture infection model. The inhibition ofC. trachomatisL2 growth was more pronounced when treated at earlier infection time points, and the removal of the inhibitors after 12 h postinfection did not rescue progeny production. Our findings revealed that altered CppA activity reduces chlamydial growth and that CppA function is likely crucial for early differentiation events. Collectively, our findings further support the importance of the protein phosphorylation network in chlamydial development.IMPORTANCEChlamydiais a significant cause of disease in humans, including sexually transmitted infections, the ocular infection trachoma, and pneumonia. Despite the critical roles of protein phosphatases in bacterial physiology, their function in pathogenesis is less clear. Our findings demonstrate that CppA, a broad-specificity type 2C protein phosphatase (PP2C), is critical for chlamydial development and further substantiate reversible phosphorylation as a key regulatory mechanism inChlamydia. Additionally, our work highlights the potential of CppA to serve as a novel target for future therapeutic strategies and supports the feasibility of designing more potent PP2C phosphatase inhibitors forChlamydiaand other pathogenic bacteria.

scholarly journals In Vitro Activity of Lefamulin against Sexually Transmitted Bacterial Pathogens

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Susanne Paukner ◽  
Astrid Gruss ◽  
Jørgen Skov Jensen
Keyword(s):  
Chlamydia Trachomatis ◽  
Bacterial Pathogens ◽  
Mycoplasma Genitalium ◽  
Standard Of Care ◽  
Multidrug Resistant ◽  
In Vitro Activity ◽  
First Line ◽  
Content Type ◽  
Sexually Transmitted

ABSTRACT The pleuromutilin antibiotic lefamulin demonstrated in vitro activity against the most relevant bacterial pathogens causing sexually transmitted infections (STI), including Chlamydia trachomatis (MIC 50/90 , 0.02/0.04 mg/liter; n = 15), susceptible and multidrug-resistant Mycoplasma genitalium (MIC range, 0.002 to 0.063 mg/liter; n = 6), and susceptible and resistant Neisseria gonorrhoeae (MIC 50/90 , 0.12/0.5 mg/liter; n = 25). The results suggest that lefamulin could be a promising first-line antibiotic for the treatment of STI, particularly in populations with high rates of resistance to standard-of-care antibiotics.

scholarly journals Type II Secretion-Dependent Aminopeptidase LapA and Acyltransferase PlaC Are Redundant for Nutrient Acquisition duringLegionella pneumophilaIntracellular Infection of Amoebas

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
pp. e00528-18 ◽  
Author(s):  
Richard C. White ◽  
Felizza F. Gunderson ◽  
Jessica Y. Tyson ◽  
Katherine H. Richardson ◽  
Theo J. Portlock ◽  
...  
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Transcriptional Analysis ◽  
Type Ii Secretion ◽  
Major Advance ◽  
Type Ii ◽  
Content Type ◽  
Broad Substrate Specificity ◽  
First Time

ABSTRACTLegionella pneumophilagenes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection ofAcanthamoeba castellanii, although these genes had been considered dispensable on the basis of the behavior of mutants lacking eitherlapAandlapBorplaC. AplaCmutant showed even higher levels oflapAandlapBtranscripts, and alapA lapBmutant showed heightening ofplaCmRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found thatlapA plaCmutants have an ~50-fold defect during infection ofA. castellanii. These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of thelapA plaCmutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection ofA. castellanii. AlapA plaC nttDmutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside theLegionellagenus. LapA was notable for having as its closest homologue anA. castellaniiprotein.IMPORTANCETransmission ofL. pneumophilato humans is facilitated by its ability to grow inAcanthamoebaspecies. We previously documented that type II secretion (T2S) promotesL. pneumophilainfection ofA. castellanii. Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection ofA. castellanii. Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within theLegionellagenus in unique ways. The results of this study represent a major advance in understandingLegionellaecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism.

scholarly journals Systematic Analysis of c-di-GMP Signaling Mechanisms and Biological Functions in Dickeya zeae EC1

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Yufan Chen ◽  
Jianuan Zhou ◽  
Mingfa Lv ◽  
Zhibin Liang ◽  
Matthew R. Parsek ◽  
...  
Keyword(s):  
Caulobacter Crescentus ◽  
Bacterial Species ◽  
Economic Losses ◽  
Signaling Proteins ◽  
Dependent Manner ◽  
Content Type ◽  
Bacterial Physiology ◽  
Diguanylate Cyclases ◽  
Genes Encoding ◽  
Rice Plantations

ABSTRACT Dickeya zeae is an important and aggressive bacterial phytopathogen that can cause substantial economic losses in banana and rice plantations. We previously showed that c-di-GMP signaling proteins (cyclases/phosphodiesterases) in D. zeae strain EC1 play a significant role in the bacterial sessile-to-motile transition. To determine whether there is any synergistic effect among these c-di-GMP signaling proteins, we prepared a series of mutant strains by generating consecutive in-frame deletions of the genes encoding diguanylate cyclases (which make c-di-GMP) and phosphodiesterases (which break down c-di-GMP), respectively, using EC1 as a parental strain. The results showed that the complete deletion of all the putative diguanylate cyclases resulted in significantly increased bacterial motility and abrogated biofilm formation but did not appear to affect pathogenicity and virulence factor production. In contrast, the deletion of all the c-di-GMP phosphodiesterase genes disabled motility and prevented the invasion of EC1 into rice seeds. By measuring the c-di-GMP concentrations and swimming motility of all the mutants, we propose that c-di-GMP controlled swimming behavior through a multitiered program in a c-di-GMP concentration-dependent manner, which could be described as an L-shaped regression curve. These features are quite different from those that have been shown for other bacterial species such as Salmonella and Caulobacter crescentus. Further analysis identified three c-di-GMP signaling proteins, i.e., PDE10355, DGC14945, and PDE14950, that play dominant roles in influencing the global c-di-GMP pool of strain EC1. The findings from this study highlight the complexity and plasticity of c-di-GMP regulatory circuits in different bacterial species. IMPORTANCE Dickeya zeae is the etiological agent of bacterial foot rot disease, which can cause massive economic losses in banana and rice plantations. Genome sequence analysis showed that D. zeae strain EC1 contains multiple c-di-GMP turnover genes, but their roles and regulatory mechanisms in bacterial physiology and virulence remain vague. By generating consecutive in-frame deletion mutants of the genes encoding c-di-GMP biosynthesis and degradation, respectively, we analyzed the individual and collective impacts of these c-di-GMP metabolic genes on the c-di-GMP global pool, bacterial physiology, and virulence. The significance of our study is in identifying the mechanism of c-di-GMP signaling in strain EC1 more clearly, which expands the c-di-GMP regulating patterns in Gram-negative species. The methods and experimental designs in this research will provide a valuable reference for the exploration of the complex c-di-GMP regulation mechanisms in other bacteria.

Effects of okadaic acid and intracellular Cl- on Na(+)-K(+)-Cl- cotransport

1995 ◽  
Vol 269 (4) ◽  
pp. C878-C883 ◽  
Author(s):  
A. A. Altamirano ◽  
G. E. Breitwieser ◽  
J. M. Russell
Keyword(s):  
Phosphatase Activity ◽  
Okadaic Acid ◽  
Protein Phosphatase ◽  
Giant Axon ◽  
Subsequent Treatment ◽  
Squid Giant Axon ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Intracellular Atp ◽  
Phosphatase Inhibitor

The Na(+)-K(+)-Cl- cotransporter of the squid giant axon requires ATP and is inhibited by intracellular Cl- (Cli-) in a concentration-dependent manner ([Cl-]i > or = 200 mM completely inhibits the cotransporter). In the present study we address the question of whether inhibition of cotransport by Cli- is due to a Cl(i-)-induced increase of protein phosphatase activity. Intracellular dialysis was used to apply the phosphatase inhibitor okadaic acid (OKA) under conditions of [Cl-]i at 0, 150, or 300 mM during measurement of cotransporter-mediated unidirectional Cl- influx into axons. At 0 mM [Cl-]i, the application of 250 nM OKA had no effect on the cotransport-mediated Cl- influx when axons were dialyzed with the normal intracellular ATP concentration ([ATP]i = 4 mM). Reduction of [ATP] to 50 microM resulted in a significant decrease of the bumetanide-sensitive CL- influx, which could be partially reversed by OKA treatment. Similarly, in ATP-limited axons with [Cl-]i at 150 mM, cotransporter influx was partially stimulated by treatment with OKA. However, axons dialyzed with 300 mM [Cl-]i ([ATP]i = 50 microM) had no measurable cotransport influx, nor was subsequent treatment with OKA able to induce a cotransport-mediated Cl- influx. We conclude that the inhibition of cotransport caused by Cli- is not the result of an increase in the OKA-sensitive protein phosphatase activity.

scholarly journals Biochemical Characterization of the POM-1 Metallo-β-Lactamase from Pseudomonas otitidis

2014 ◽  
Vol 59 (3) ◽  
pp. 1755-1758 ◽  
Author(s):  
Luisa Borgianni ◽  
Filomena De Luca ◽  
Maria Cristina Thaller ◽  
Yunsop Chong ◽  
Gian Maria Rossolini ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Substrate Specificity ◽  
Biochemical Characterization ◽  
Content Type ◽  
Broad Substrate Specificity ◽  
Catalytic Activities

ABSTRACTThe POM-1 metallo-β-lactamase is a subclass B3 resident enzyme produced byPseudomonas otitidis, a pathogen causing otic infections. The enzyme was overproduced inEscherichia coliBL21(DE3), purified by chromatography, and subjected to structural and functional analysis. The purified POM-1 is a tetrameric enzyme of broad substrate specificity with higher catalytic activities with penicillins and carbapenems than with cephalosporins.

scholarly journals Corynebacterium diphtheriaeIron-Regulated Surface Protein HbpA Is Involved in the Utilization of the Hemoglobin-Haptoglobin Complex as an Iron Source

2018 ◽  
Vol 200 (7) ◽  
Author(s):  
Lindsey R. Lyman ◽  
Eric D. Peng ◽  
Michael P. Schmitt
Keyword(s):  
Sequence Similarity ◽  
Bacterial Pathogens ◽  
Surface Protein ◽  
Surface Proteins ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Human Respiratory Tract ◽  
Homologous Proteins ◽  
Content Type ◽  
Iron Source

ABSTRACTCorynebacterium diphtheriaeutilizes various heme-containing proteins, including hemoglobin (Hb) and the hemoglobin-haptoglobin complex (Hb-Hp), as iron sources during growth in iron-depleted environments. The ability to utilize Hb-Hp as an iron source requires the surface-anchored proteins HtaA and either ChtA or ChtC. The ability to bind hemin, Hb, and Hb-Hp by each of theseC. diphtheriaeproteins requires the previously characterized conserved region (CR) domain. In this study, we identified an Hb-Hp binding protein, HbpA (38.5 kDa), which is involved in the acquisition of hemin iron from Hb-Hp. HbpA was initially identified from total cell lysates as an iron-regulated protein that binds to both Hb and Hb-Hpin situ. HbpA does not contain a CR domain and has sequence similarity only to homologous proteins present in a limited number ofC. diphtheriaestrains. Transcription ofhbpAis regulated in an iron-dependent manner that is mediated by DtxR, a global iron-dependent regulator. Deletion ofhbpAfromC. diphtheriaeresults in a reduced ability to utilize Hb-Hp as an iron source but has little or no effect on the ability to use Hb or hemin as an iron source. Cell fractionation studies showed that HbpA is both secreted into the culture supernatant and associated with the membrane, where its exposure on the bacterial surface allows HbpA to bind Hb and Hb-Hp. The identification and analysis of HbpA enhance our understanding of iron uptake inC. diphtheriaeand indicate that the acquisition of hemin iron from Hb-Hp may involve a complex mechanism that requires multiple surface proteins.IMPORTANCEThe ability to utilize host iron sources, such as heme and heme-containing proteins, is essential for many bacterial pathogens to cause disease. In this study, we have identified a novel factor (HbpA) that is crucial for the use of hemin iron from the hemoglobin-haptoglobin complex (Hb-Hp). Hb-Hp is considered one of the primary sources of iron for certain bacterial pathogens. HbpA has no similarity to the previously identified Hb-Hp binding proteins, HtaA and ChtA/C, and is found only in a limited group ofC. diphtheriaestrains. Understanding the function of HbpA may significantly increase our knowledge of how this important human pathogen can acquire host iron that allows it to survive and cause disease in the human respiratory tract.

scholarly journals Effect of Phosphatase Activity of the Control of Virulence Sensor (CovS) on Clindamycin-Mediated Streptolysin O Production in Group A Streptococcus

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Chuan Chiang-Ni ◽  
Huei-Chuan Tseng ◽  
Yong-An Shi ◽  
Cheng-Hsun Chiu
Keyword(s):  
Phosphatase Activity ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Tissue Destruction ◽  
Synthesis Inhibitor ◽  
Content Type ◽  
Streptolysin O ◽  
Group A

ABSTRACT Severe manifestations of group A Streptococcus (GAS) infections are associated with massive tissue destruction and high mortality. Clindamycin (CLI), a bacterial protein synthesis inhibitor, is recommended for treating patients with severe invasive GAS infection. Nonetheless, the subinhibitory concentration of CLI induces the production of GAS virulent exoproteins, such as streptolysin O (SLO) and NADase, which would enhance bacterial virulence and invasiveness. A better understanding of the molecular mechanism of how CLI triggers GAS virulence factor expression will be critical to develop appropriate therapeutic approaches. The present study shows that CLI activates SLO and NADase expressions in the emm1-type CLI-susceptible wild-type strain but not in covS or control of virulence sensor (CovS) phosphatase-inactivated mutants. Supplementation with Mg2+, which is a CovS phosphatase inhibitor, inhibits the CLI-mediated SLO upregulation in a dose-dependent manner in CLI-susceptible and CLI-resistant strains. These results not only reveal that the phosphorylation of response regulator CovR is essential for responding to CLI stimuli, but also suggest that inhibiting the phosphatase activity of CovS could be a potential strategy for the treatment of invasive GAS infection with CLI.

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