scholarly journals A Trap-Door Mechanism for Zinc Acquisition by Streptococcus pneumoniae AdcA

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhenyao Luo ◽  
Jacqueline R. Morey ◽  
Evelyne Deplazes ◽  
Alina Motygullina ◽  
Aimee Tan ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Binding Site ◽  
Essential Element ◽  
Zinc Binding ◽  
Surface Element ◽  
Dynamic Surface ◽  
Content Type ◽  
Trap Door ◽  
Functional Basis ◽  
Loop Region

ABSTRACT Zinc is an essential element in all domains of life. Nonetheless, how prokaryotes achieve selective acquisition of zinc from the extracellular environment remains poorly understood. Here, we elucidate a novel mechanism for zinc-binding in AdcA, a solute-binding protein of Streptococcus pneumoniae. Crystal structure analyses reveal the two-domain organization of the protein and show that only the N-terminal domain (AdcAN) is necessary for zinc import. Zinc binding induces only minor changes in the global protein conformation of AdcA and stabilizes a highly mobile loop within the AdcAN domain. This loop region, which is conserved in zinc-specific solute-binding proteins, facilitates closure of the AdcAN binding site and is crucial for zinc acquisition. Collectively, these findings elucidate the structural and functional basis of selective zinc uptake in prokaryotes. IMPORTANCE Zinc is an essential nutrient for the virulence of bacterial pathogens such as Streptococcus pneumoniae. Many Gram-positive bacteria use a two-domain lipoprotein for zinc acquisition, but how this class of metal-recruiting proteins acquire zinc and interact with the uptake machinery has remained poorly defined. We report the first structure of a two-domain lipoprotein, AdcA from S. pneumoniae, and use computational, spectroscopic, and microbiological approaches to provide new insights into the functional basis of zinc recruitment. Our findings reveal that AdcA employs a novel mechanism for zinc binding that we have termed the “trap-door” mechanism, and we show how the static metal-binding site of the protein, which confers its selectivity for zinc ions, is combined with a dynamic surface element to facilitate zinc recruitment and import into the bacterium. Together, these findings expand our understanding of how bacteria acquire zinc from the environment and provide a foundation for inhibiting this process, through antimicrobial targeting of the dynamic structural elements to block bacterial zinc scavenging.

scholarly journals The stability of HIV-2 Vpx and Vpr proteins is regulated by the presence or absence of zinc-binding sites and poly-proline motifs with distinct roles

2020 ◽  
Vol 101 (9) ◽  
pp. 997-1007
Author(s):  
Kazunori Shimagaki ◽  
Ryoko Koga ◽  
Haruna Fujino ◽  
Ami Ahagon ◽  
Hiroshi Tateishi ◽  
...  
Keyword(s):  
Binding Site ◽  
Zinc Binding ◽  
Hydrophobic Region ◽  
Unique Region ◽  
Content Type ◽  
C Terminus ◽  
Immunodeficiency Virus ◽  
Zinc Binding Site ◽  
The Stability

The Vpx and Vpr proteins of human immunodeficiency virus type 2 (HIV-2) are important for virus replication. Although these proteins are homologous, Vpx is expressed at much higher levels than Vpr. Previous studies demonstrated that this difference results from the presence of an HHCC zinc-binding site in Vpx that is absent in Vpr. Vpx has another unique region, a poly-proline motif (PPM) of seven consecutive prolines at the C-terminus. Using PPM point mutants of Vpx, this study demonstrated that these seven consecutive prolines are critical for suppressing proteasome degradation of Vpx in the absence of Gag. Both the PPM and the zinc-binding site stabilize Vpx but do so via different mechanisms. PPM and zinc-binding site mutants overexpressed in Escherichia coli aggregated readily, indicating that these motifs normally prevent exposure of the hydrophobic region outside the structure. Furthermore, introduction of the zinc-binding site and the PPM into Vpr increased the level of Vpr expression so that it was as high as that of Vpx. Intriguingly, HIV-2 has evolved to express Vpx at high levels and Vpr at low levels based on the presence and absence of these two motifs with distinct roles.

scholarly journals Antibodies to Streptococcus pneumoniae Capsular Polysaccharide Enhance Pneumococcal Quorum Sensing

mBio ◽  
2011 ◽  
Vol 2 (5) ◽  
Author(s):  
Masahide Yano ◽  
Shruti Gohil ◽  
J. Robert Coleman ◽  
Catherine Manix ◽  
Liise-anne Pirofski
Keyword(s):  
Monoclonal Antibodies ◽  
Streptococcus Pneumoniae ◽  
Quorum Sensing ◽  
Direct Effect ◽  
Pneumococcal Disease ◽  
Effector Cell ◽  
Capsular Polysaccharide ◽  
Genetic Exchange ◽  
Content Type ◽  
Specific Antibodies

ABSTRACTThe use of pneumococcal capsular polysaccharide (PPS)-based vaccines has resulted in a substantial reduction in invasive pneumococcal disease. However, much remains to be learned about vaccine-mediated immunity, as seven-valent PPS-protein conjugate vaccine use in children has been associated with nonvaccine serotype replacement and 23-valent vaccine use in adults has not prevented pneumococcal pneumonia. In this report, we demonstrate that certain PPS-specific monoclonal antibodies (MAbs) enhance the transformation frequency of two differentStreptococcus pneumoniaeserotypes. This phenomenon was mediated by PPS-specific MAbs that agglutinate but do not promote opsonic effector cell killing of the homologous serotypeinvitro. Compared to the autoinducer, competence-stimulating peptide (CSP) alone, transcriptional profiling of pneumococcal gene expression after incubation with CSP and one such MAb to the PPS of serotype 3 revealed changes in the expression of competence (com)-related and bacteriocin-like peptide (blp) genes involved in pneumococcal quorum sensing. This MAb was also found to induce a nearly 2-fold increase in CSP2-mediated bacterial killing or fratricide. These observations reveal a novel, direct effect of PPS-binding MAbs on pneumococcal biology that has important implications for antibody immunity to pneumococcus in the pneumococcal vaccine era. Taken together, our data suggest heretofore unsuspected mechanisms by which PPS-specific antibodies could affect genetic exchange and bacterial viability in the absence of host cells.IMPORTANCECurrent thought holds that pneumococcal capsular polysaccharide (PPS)-binding antibodies protect against pneumococcus by inducing effector cell opsonic killing of the homologous serotype. While such antibodies are an important part of how pneumococcal vaccines protect against pneumococcal disease, PPS-specific antibodies that do not exhibit this activity but are highly protective against pneumococcus in mice have been identified. This article examines the effect of nonopsonic PPS-specific monoclonal antibodies (MAbs) on the biology ofStreptococcus pneumoniae. The results showed that in the presence of a competence-stimulating peptide (CSP), such MAbs increase the frequency of pneumococcal transformation. Further studies with one such MAb showed that it altered the expression of genes involved in quorum sensing and increased competence-induced killing or fratricide. These findings reveal a novel, previously unsuspected mechanism by which certain PPS-specific antibodies exert a direct effect on pneumococcal biology that has broad implications for bacterial clearance, genetic exchange, and antibody immunity to pneumococcus.

scholarly journals The Structure of the Cysteine-Rich Domain of Plasmodium falciparum P113 Identifies the Location of the RH5 Binding Site

mBio ◽  
2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Ivan Campeotto ◽  
Francis Galaway ◽  
Shahid Mehmood ◽  
Lea K. Barfod ◽  
Doris Quinkert ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Binding Site ◽  
Structural Information ◽  
Blood Stage ◽  
Site Directed Mutagenesis ◽  
Effective Vaccine ◽  
Binding Region ◽  
Fab Fragment ◽  
Content Type ◽  
Blood Stage Malaria

ABSTRACT Plasmodium falciparum RH5 is a secreted parasite ligand that is essential for erythrocyte invasion through direct interaction with the host erythrocyte receptor basigin. RH5 forms a tripartite complex with two other secreted parasite proteins, CyRPA and RIPR, and is tethered to the surface of the parasite through membrane-anchored P113. Antibodies against RH5, CyRPA, and RIPR can inhibit parasite invasion, suggesting that vaccines containing these three components have the potential to prevent blood-stage malaria. To further explore the role of the P113-RH5 interaction, we selected monoclonal antibodies against P113 that were either inhibitory or noninhibitory for RH5 binding. Using a Fab fragment as a crystallization chaperone, we determined the crystal structure of the RH5 binding region of P113 and showed that it is composed of two domains with structural similarities to rhamnose-binding lectins. We identified the RH5 binding site on P113 by using a combination of hydrogen-deuterium exchange mass spectrometry and site-directed mutagenesis. We found that a monoclonal antibody to P113 that bound to this interface and inhibited the RH5-P113 interaction did not inhibit parasite blood-stage growth. These findings provide further structural information on the protein interactions of RH5 and will be helpful in guiding the development of blood-stage malaria vaccines that target RH5. IMPORTANCE Malaria is a deadly infectious disease primarily caused by the parasite Plasmodium falciparum. It remains a major global health problem, and there is no highly effective vaccine. A parasite protein called RH5 is centrally involved in the invasion of host red blood cells, making it—and the other parasite proteins it interacts with—promising vaccine targets. We recently identified a protein called P113 that binds RH5, suggesting that it anchors RH5 to the parasite surface. In this paper, we use structural biology to locate and characterize the RH5 binding region on P113. These findings will be important to guide the development of new antimalarial vaccines to ultimately prevent this disease, which affects some of the poorest people on the planet.

scholarly journals Nosocomial Outbreak of Drug-Resistant Streptococcus pneumoniae Serotype 9V in an Adult Respiratory Medicine Ward

2016 ◽  
Vol 55 (3) ◽  
pp. 776-782 ◽  
Author(s):  
Elita Jauneikaite ◽  
Zareena Khan-Orakzai ◽  
Georgia Kapatai ◽  
Susannah Bloch ◽  
Julie Singleton ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Lower Respiratory Tract ◽  
Sequence Type ◽  
Resistance Pattern ◽  
Whole Genome ◽  
Drug Resistant ◽  
Nosocomial Outbreak ◽  
Content Type ◽  
Rapid Transmission ◽  
Case Presentations

ABSTRACT Streptococcus pneumoniae infections arising in hospitalized patients are often assumed to be sporadic and linked to community acquisition. Here, whole-genome sequencing was used to demonstrate nosocomial acquisition of antimicrobial-resistant sequence type 156 (ST156) serotype 9V S. pneumoniae in 3 respiratory patients that resulted in two bacteremias and one lower respiratory tract infection. Two of the cases arose in patients who had recently been discharged from the hospital and were readmitted from the community. Nosocomial spread was suspected solely because of the highly unusual resistance pattern and case presentations within 24 h of one another. The outbreak highlights the potential for rapid transmission and the short incubation period in the respiratory ward setting.

scholarly journals Involvement of RpoN in Regulating Bacterial Arsenite Oxidation

2012 ◽  
Vol 78 (16) ◽  
pp. 5638-5645 ◽  
Author(s):  
Yoon-Suk Kang ◽  
Brian Bothner ◽  
Christopher Rensing ◽  
Timothy R. McDermott
Keyword(s):  
Binding Site ◽  
Sigma Factor ◽  
Model Organism ◽  
Coding Region ◽  
Rt Pcr ◽  
Obvious Effect ◽  
Content Type ◽  
Definitive Evidence ◽  
Relative Contribution ◽  
Insertional Inactivation

ABSTRACTIn this study with the model organismAgrobacterium tumefaciens, we used a combination oflacZgene fusions, reverse transcriptase PCR (RT-PCR), and deletion and insertional inactivation mutations to show unambiguously that the alternative sigma factor RpoN participates in the regulation of AsIIIoxidation. A deletion mutation that removed the RpoN binding site from theaioBApromoter and anaacC3(gentamicin resistance) cassette insertional inactivation of therpoNcoding region eliminatedaioBAexpression and AsIIIoxidation, althoughrpoNexpression was not related to cell exposure to AsIII. Putative RpoN binding sites were identified throughout the genome and, as examples, included promoters foraioB,phoB1,pstS1,dctA,glnA,glnB, andflgBthat were examined by using qualitative RT-PCR andlacZreporter fusions to assess the relative contribution of RpoN to their transcription. The expressions ofaioBanddctAin the wild-type strain were considerably enhanced in cells exposed to AsIII, and both genes were silent in therpoN::aacC3mutant regardless of AsIII. The expression level ofglnAwas not influenced by AsIIIbut was reduced (but not silent) in therpoN::aacC3mutant and further reduced in the mutant under N starvation conditions. TherpoN::aacC3mutation had no obvious effect on the expression ofglnB,pstS1,phoB1, orflgB. These experiments provide definitive evidence to document the requirement of RpoN for AsIIIoxidation but also illustrate that the presence of a consensus RpoN binding site does not necessarily link the associated gene with regulation by AsIIIor by this sigma factor.

scholarly journals Protection against Streptococcus pneumoniae Invasive Pathogenesis by a Protein-Based Vaccine Is Achieved by Suppression of Nasopharyngeal Bacterial Density during Influenza A Virus Coinfection

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
M. Nadeem Khan ◽  
Qingfu Xu ◽  
Michael E. Pichichero
Keyword(s):  
T Cells ◽  
Streptococcus Pneumoniae ◽  
Influenza A Virus ◽  
Influenza A ◽  
Passive Transfer ◽  
Bacterial Density ◽  
Content Type ◽  
Adult Mice ◽  
Significant Difference ◽  
Colonization Density

ABSTRACTAn increase inStreptococcus pneumoniaenasopharynx (NP) colonization density during a viral coinfection initiates pathogenesis. To mimic naturalS. pneumoniaepathogenesis, we commensally colonized the NPs of adult C57BL/6 mice withS. pneumoniaeserotype (ST) 6A or 8 and then coinfected them with mouse-adapted H1N1 influenza A virus (PR/8/34).S. pneumoniaeestablished effective commensal colonization, and influenza virus coinfection causedS. pneumoniaeNP density to increase, resulting in bacteremia and mortality. We then studied histidine triad protein D (PhtD), anS. pneumoniaeadhesin vaccine candidate, for its ability to prevent invasiveS. pneumoniaedisease in adult and infant mice. In adult mice, the efficacy of PhtD vaccination was compared with that of PCV13. Vaccination with PCV13 led to a greater reduction ofS. pneumoniaeNP density (>2.5 log units) than PhtD vaccination (∼1-log-unit reduction). However, no significant difference was observed with regard to the prevention ofS. pneumoniaebacteremia, and there was no difference in mortality. Depletion of CD4+T cells in PhtD-vaccinated adult mice, but not PCV13-vaccinated mice, caused a loss of vaccine-induced protection. In infant mice, passive transfer of antisera or CD4+T cells from PhtD-vaccinated adult mice led to a nonsignificant reduction in NP colonization density, whereas passive transfer of antisera and CD4+T cells was needed to cause a significant reduction in NP colonization density. For the first time, these data show an outcome with regard to prevention of invasiveS. pneumoniaepathogenesis with a protein vaccine similar to that which occurs with a glycoconjugate vaccine despite a less robust reduction in NP bacterial density.

scholarly journals Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae

mBio ◽  
2014 ◽  
Vol 5 (1) ◽  
Author(s):  
Peter Mellroth ◽  
Tatyana Sandalova ◽  
Alexey Kikhney ◽  
Francisco Vilaplana ◽  
Dusan Hesek ◽  
...  
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Solution Structure ◽  
Substrate Binding ◽  
Substrate Recognition ◽  
Lytic Activity ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Content Type ◽  
Peptidoglycan Synthesis

ABSTRACT The cytosolic N-acetylmuramoyl-l-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or β-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-Å crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

scholarly journals Acute Mastoiditis in the Pneumococcal Conjugate Vaccine Era

2014 ◽  
Vol 21 (8) ◽  
pp. 1189-1191 ◽  
Author(s):  
Sharon Ovnat Tamir ◽  
Yehudah Roth ◽  
Ilan Dalal ◽  
Abraham Goldfarb ◽  
Tal Marom
Keyword(s):  
Streptococcus Pneumoniae ◽  
Middle Ear ◽  
Pneumococcal Conjugate Vaccine ◽  
Inverse Relationship ◽  
Acute Otitis Media ◽  
Acute Mastoiditis ◽  
Conjugate Vaccines ◽  
Content Type ◽  
Bacterial Cultures ◽  
Middle Ear Fluid

ABSTRACTFollowing the introduction of the 7- and 13-valent pneumococcal conjugate vaccines, we observed an inverse relationship between the increasing rate of immunized children and the proportion of middle ear fluid cultures collected during acute mastoiditis episodes that tested positive forStreptococcus pneumoniaeamong a subset of children 0 to 6 years old who had initially presented with severe acute otitis media and had bacterial cultures collected during tympanocentesis or from spontaneous otorrhea.

scholarly journals Topology of Streptococcus pneumoniae CpsC, a Polysaccharide Copolymerase and Bacterial Protein Tyrosine Kinase Adaptor Protein

2014 ◽  
Vol 197 (1) ◽  
pp. 120-127 ◽  
Author(s):  
Jonathan J. Whittall ◽  
Renato Morona ◽  
Alistair J. Standish
Keyword(s):  
Streptococcus Pneumoniae ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Adaptor Protein ◽  
Tyrosine Kinase Activity ◽  
Content Type ◽  
Protein Tyrosine ◽  
C Terminus

In Gram-positive bacteria, tyrosine kinases are split into two proteins, the cytoplasmic tyrosine kinase and a transmembrane adaptor protein. InStreptococcus pneumoniae, this transmembrane adaptor is CpsC, with the C terminus of CpsC critical for interaction and subsequent tyrosine kinase activity of CpsD. Topology predictions suggest that CpsC has two transmembrane domains, with the N and C termini present in the cytoplasm. In order to investigate CpsC topology, we used a chromosomal hemagglutinin (HA)-tagged Cps2C protein inS. pneumoniaestrain D39. Incubation of both protoplasts and membranes with carboxypeptidase B (CP-B) resulted in complete degradation of HA-Cps2C in all cases, indicating that the C terminus of Cps2C was likely extracytoplasmic and hence that the protein's topology was not as predicted. Similar results were seen with membranes fromS. pneumoniaestrain TIGR4, indicating that Cps4C also showed similar topology. A chromosomally encoded fusion of HA-Cps2C and Cps2D was not degraded by CP-B, suggesting that the fusion fixed the C terminus within the cytoplasm. However, capsule synthesis was unaltered by this fusion. Detection of the CpsC C terminus by flow cytometry indicated that it was extracytoplasmic in approximately 30% of cells. Interestingly, a mutant in the protein tyrosine phosphatase CpsB had a significantly greater proportion of positive cells, although this effect was independent of its phosphatase activity. Our data indicate that CpsC possesses a varied topology, with the C terminus flipping across the cytoplasmic membrane, where it interacts with CpsD in order to regulate tyrosine kinase activity.

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