Evolution of Gram+ Streptococcus pyogenes has maximized efficiency of the Sortase A cleavage site

Human plasminogen (hPg)-binding M-protein (PAM), a major virulence factor of Pattern D Streptococcus pyogenes (GAS), is the primary receptor responsible for binding and activating hPg. PAM is covalently bound to the cell wall (CW) through cell membrane (CM)-resident sortase A (SrtA)-catalyzed cleavage of the PAM-proximal C-terminal LPST¯-GEAA motif present immediately upstream of its transmembrane domain (TMD), and subsequent transpeptidation to the CW. These steps expose the N-terminus of PAM to the extracellular milieu (EM) to interact with PAM ligands, e.g., hPg. Previously, we found that inactivation of SrtA showed little reduction in functional binding of PAM to hPg, indicating that PAM retained in the cell membrane (CM) by the TMD nonetheless exposed its N-terminus to the EM. In the current study, we assessed the effects of mutating the Thr4 (P1) residue of the SrtA-cleavage site in PAM (Thr355 in PAM) to delay PAM in the CM in the presence of SrtA. Using rSrtA in vitro, LPSYGEAA and LPSWGEAA peptides were shown to have low activities, while LPSTGEAA had the highest activity. Isolated CM fractions of AP53/DSrtA cells showed that LPSYGEAA and LPSWGEAA peptides were cleaved at substantially faster rates than LPSTGEAA, even in CMs with an AP53/DSrtA/PAM[T355Y] double mutation, but the transpeptidation step did not occur. These results implicate another CM-resident enzyme that cleaves LPSYGEAA and LPSWGEAA motifs, most likely LPXTGase, but cannot catalyze the transpeptidation step. We conclude that the natural P1 (Thr) of the SrtA cleavage site has evolved to dampen PAM from nonfunctional cleavage by LPXTGase.

Download Full-text

Effects of l- and d-REKR amino acid-containing peptides on HIV and SIV envelope glycoprotein precursor maturation and HIV and SIV replication

Biochemical Journal ◽

10.1042/bj20020052 ◽

2002 ◽

Vol 366 (3) ◽

pp. 863-872 ◽

Cited By ~ 4

Author(s):

Bouchaib BAHBOUHI ◽

Nathalie CHAZAL ◽

Nabil Georges SEIDAH ◽

Cristina CHIVA ◽

Marcelo KOGAN ◽

...

Keyword(s):

Amino Acid ◽

Envelope Glycoprotein ◽

Cleavage Site ◽

Molecular Level ◽

Principal Cell ◽

Inhibition Constant ◽

Glycoprotein Precursor ◽

C Terminus ◽

N Terminus

The aim of the present study was to evaluate the capacity of synthetic l- and d-peptides encompassing the HIV-1BRU gp160 REKR cleavage site to interfere with HIV and simian immuno-deficiency virus (SIV) replication and maturation of the envelope glycoprotein (Env) precursors. To facilitate their penetration into cells, a decanoyl (dec) group was added at the N-terminus. The sequences synthesized included dec5d or dec5l (decREKRV), dec9d or dec9l (decRVVQREKRV) and dec14d or dec14l (TKAKRRVVQREKRV). The peptide dec14d was also prepared with a chloromethane (cmk) group as C-terminus. Because l-peptides exhibit significant cytotoxicity starting at 35μM, further characterization was conducted mostly with d-peptides, which exhibited no cytotoxicity at concentrations higher than 70μM. The data show that only dec14d and dec14dcmk could inhibit HIV-1BRU, HIV-2ROD and SIVmac251 replication and their syncytium-inducing capacities. Whereas peptides dec5d and dec9d were inactive, dec14dcmk was at least twice as active as peptide dec14d. At the molecular level, our data show a direct correlation between anti-viral activity and the ability of the peptides to interfere with maturation of the Env precursors. Furthermore, we show that when tested in vitro the dec14d peptide inhibited PC7 with an inhibition constant Ki = 4.6μM, whereas the peptide dec14l preferentially inhibited furin with a Ki = 28μM. The fact that PC7 and furin are the major prohormone convertases reported to be expressed in T4 lymphocytes, the principal cell targets of HIV, suggests that they are involved in the maturation of HIV and SIV Env precursors.

Download Full-text

Carvacrol exhibits rapid bactericidal activity against Streptococcus pyogenes through cell membrane damage

Scientific Reports ◽

10.1038/s41598-020-79713-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Niluni M. Wijesundara ◽

Song F. Lee ◽

Zhenyu Cheng ◽

Ross Davidson ◽

H. P. Vasantha Rupasinghe

Keyword(s):

Cell Membrane ◽

Streptococcus Pyogenes ◽

Bactericidal Activity ◽

Membrane Damage ◽

Membrane Integrity ◽

Streptococcal Pharyngitis ◽

Cell Membrane Integrity ◽

Cell Membrane Damage ◽

Antibiotic Development

AbstractStreptococcus pyogenes is an important human pathogen worldwide. The identification of natural antibacterial phytochemicals has renewed interest due to the current scarcity of antibiotic development. Carvacrol is a monoterpenoid found in herbs. We evaluated carvacrol alone and combined with selected antibiotics against four strains of S. pyogenes in vitro. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvacrol against S. pyogenes were 125 µg/mL (0.53 mM) and 250 µg/mL (1.05 mM), respectively. Kill curve results showed that carvacrol exhibits instantaneous bactericidal activity against S. pyogenes. We also demonstrated the potential mechanism of action of carvacrol through compromising the cell membrane integrity. Carvacrol induced membrane integrity changes leading to leakage of cytoplasmic content such as lactate dehydrogenase enzymes and nucleic acids. We further confirmed dose-dependent rupturing of cells and cell deaths using transmission electron microscopy. The chequerboard assay results showed that carvacrol possesses an additive-synergistic effect with clindamycin or penicillin. Carvacrol alone, combined with clindamycin or penicillin, can be used as a safe and efficacious natural health product for managing streptococcal pharyngitis.

Download Full-text

A conserved Guided Entry of Tail-anchored pathway is involved in the trafficking of tail-anchored membrane proteins in Plasmodium falciparum.

10.1101/2021.05.03.442402 ◽

2021 ◽

Author(s):

Tarkeshwar Kumar ◽

Satarupa Maitra ◽

Abdur Rahman ◽

Souvik Bhattacharjee

Keyword(s):

Plasmodium Falciparum ◽

Targeted Delivery ◽

Transmembrane Domain ◽

Protein Translocation ◽

Signal Sequence ◽

Association Studies ◽

C Terminus ◽

N Terminus ◽

Ta Proteins

Tail-anchored (TA) proteins are defined by the absence of N-terminus signal sequence and the presence of a single transmembrane domain (TMD) proximal to their extreme C-terminus. They play fundamental roles in cellular processes including vesicular trafficking, protein translocation and quality control. Accordingly, TA proteins are post-translationally integrated by the Guided Entry of TA (GET) pathway to the cellular membranes; with their N-terminus oriented towards the cytosol and C-terminus facing the organellar lumen. The TA repertoire and the GET machinery have been extensively characterized in the yeast and mammalian systems, however, they remain elusive in the human malaria parasite Plasmodium falciparum. In this study, we bioinformatically predicted a total of 63 TA proteins in the P. falciparum proteome and revealed the association of their subset with the P. falciparum homolog of Get3 (PfGet3). In addition, our proximity labelling studies either definitively identified or shortlisted the other eligible GET constituents, and our in vitro association studies validated associations between PfGet3 and the corresponding homologs of Get4 and Get2 in P. falciparum. Collectively, this study reveals the presence of proteins with hallmark TA signatures and the involvement of evolutionary conserved GET trafficking pathway for their targeted delivery within the parasite.

Download Full-text

A conserved guided entry of tail-anchored pathway is involved in the trafficking of a subset of membrane proteins in Plasmodium falciparum

PLoS Pathogens ◽

10.1371/journal.ppat.1009595 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1009595

Author(s):

Tarkeshwar Kumar ◽

Satarupa Maitra ◽

Abdur Rahman ◽

Souvik Bhattacharjee

Keyword(s):

Plasmodium Falciparum ◽

Targeted Delivery ◽

Transmembrane Domain ◽

Protein Translocation ◽

Signal Sequence ◽

Association Studies ◽

C Terminus ◽

N Terminus ◽

Ta Proteins

Tail-anchored (TA) proteins are defined by the absence of N-terminus signal sequence and the presence of a single transmembrane domain (TMD) proximal to their C-terminus. They play fundamental roles in cellular processes including vesicular trafficking, protein translocation and quality control. Some of the TA proteins are post-translationally integrated by the Guided Entry of TA (GET) pathway to the cellular membranes; with their N-terminus oriented towards the cytosol and C-terminus facing the organellar lumen. The TA repertoire and the GET machinery have been extensively characterized in the yeast and mammalian systems, however, they remain elusive in the human malaria parasite Plasmodium falciparum. In this study, we bioinformatically predicted a total of 63 TA proteins in the P. falciparum proteome and revealed the association of a subset with the P. falciparum homolog of Get3 (PfGet3). In addition, our proximity labelling studies either definitively identified or shortlisted the other eligible GET constituents, and our in vitro association studies validated associations between PfGet3 and the corresponding homologs of Get4 and Get2 in P. falciparum. Collectively, this study reveals the presence of proteins with hallmark TA signatures and the involvement of evolutionary conserved GET trafficking pathway for their targeted delivery within the parasite.

Download Full-text

Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro

Journal of General Virology ◽

10.1099/0022-1317-81-11-2771 ◽

2000 ◽

Vol 81 (11) ◽

pp. 2771-2781 ◽

Cited By ~ 28

Author(s):

Aiming Wang ◽

Hélène Sanfaçon

Keyword(s):

Cleavage Site ◽

Terminal Region ◽

Proteolytic Processing ◽

Site Directed Mutagenesis ◽

Cdna Clones ◽

Cleavage Sites ◽

N Terminus ◽

Partial Cdna ◽

Coding Capacity

Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domains for the putative NTP-binding protein, VPg, 3C-like protease and a putative RNA-dependent RNA polymerase in its C-terminal region. The N-terminal region of P1, with a coding capacity for a protein (or a precursor) of 67 kDa, has not been characterized. Using partial cDNA clones, it is shown that the 3C-like protease can process the N-terminal region of P1 at a novel cleavage site in vitro, allowing the release of two proteins, X1 (located at the N terminus of P1) and X2 (located immediately upstream of the NTB domain). P1 precursors in which the protease was inactive or absent were not cleaved by exogenously added protease, suggesting that P1 processing was predominantly in cis. Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q423/G and Q620/G are the X1-X2 and X2-NTB cleavage sites, respectively. The putative X1 protein contains a previously identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature protein released from the N terminus of comovirus P1 polyproteins) and to the corresponding region of other nepovirus P1 polyproteins. These results raise the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruses.

Download Full-text

The M Protein of Streptococcus pyogenes Strain AP53 Retains Cell Surface Functional Plasminogen Binding after Inactivation of the Sortase A Gene

Journal of Bacteriology ◽

10.1128/jb.00096-20 ◽

2020 ◽

Vol 202 (10) ◽

Author(s):

Brady T. Russo ◽

Yetunde A. Ayinuola ◽

Damini Singh ◽

Katelyn Carothers ◽

Vincent A. Fischetti ◽

...

Keyword(s):

Cell Wall ◽

Cell Membrane ◽

Cell Surface ◽

Streptococcus Pyogenes ◽

Cytoplasmic Membrane ◽

M Protein ◽

Sortase A ◽

Content Type ◽

M Proteins ◽

Group A

ABSTRACT Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 ΔsrtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface. IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Download Full-text

The role of glycosylation in the N-terminus of the hemagglutinin of a unique H4N2 with a natural polybasic cleavage site in virus fitness in vitro and in vivo

Virulence ◽

10.1080/21505594.2021.1881344 ◽

2021 ◽

Vol 12 (1) ◽

pp. 666-678

Author(s):

Marcel Gischke ◽

Ola Bagato ◽

Angele Breithaupt ◽

David Scheibner ◽

Claudia Blaurock ◽

...

Keyword(s):

Cleavage Site ◽

N Terminus ◽

Virus Fitness

Download Full-text

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

Biochemical Journal ◽

10.1042/bcj20190812 ◽

2019 ◽

Vol 476 (24) ◽

pp. 3835-3847 ◽

Cited By ~ 1

Author(s):

Aliyath Susmitha ◽

Kesavan Madhavan Nampoothiri ◽

Harsha Bajaj

Keyword(s):

Protein Engineering ◽

Cell Attachment ◽

Functional Characterization ◽

Protein Structures ◽

Structural Similarity ◽

Surface Proteins ◽

Sortase A ◽

Peptide Cleavage ◽

New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Download Full-text

In Vitro Studies on the Mechanism of the Antifibrino-lytic Action of E-Aminocaproic Acid (EACA)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651238 ◽

1968 ◽

Vol 19 (03/04) ◽

pp. 584-592 ◽

Cited By ~ 2

Author(s):

Hanna Lukasiewicz ◽

S Niewiarowski

Keyword(s):

Aminocaproic Acid ◽

Test Tube ◽

In Vitro Studies ◽

Lytic Action ◽

Human Plasminogen ◽

Experimental Findings ◽

Fibrin Clots

Summary and Conclusion1. It has been found that EACA does not inhibit activation of human plasminogen into plasmin by SK and UK in a concentration of 5 × 10–2 M. The activation of bovine plasminogen by SK and UK is inhibited by this concentration of EACA but not by a lower one.2. EACA in concentrations of 1,5 × 10–1 – 10–4 M does not inhibit casein proteolysis by plasmin. The proteolysis of fibrinogen and fibrin measured by the release of TCA soluble tyrosine is inhibited by EACA in concentrations of 1,5 × 10–1 – 10–2 M.3. The lysis of non-stabilized clots by plasmin measured in a test tube was inhibited by an EACA concentration of 5 × 10–3 – 5 × 10–4 M. The lysis of stabilized clots by plasmin was inhibited by an EACA concentration of 10–5 M.4. On the basis of experimental findings and data given in literature the authors postulate that the mechanism of the antifibrinolytic effects of EACA consists mainly in a modification of plasmin action on fibrin. These effects are dependent on the structure of the fibrin clots.

Download Full-text

ATIVIDADE ANTIMICROBIANA IN VITRO DO EXTRATO AQUOSO, HIDROALCOÓLICO E ALCOÓLICO DE FOLHAS DE ESPÉCIES DA FAMÍLIA LAMIACEAE

Revista Prevenção de Infecção e Saúde ◽

10.26694/repis.v4i0.7072 ◽

2018 ◽

Vol 4 ◽

Author(s):

Karlynne Freire Mendonça ◽

José Klauber Roger Carneiro ◽

Maria Auxiliadora Silva Oliveira

Keyword(s):

Staphylococcus Aureus ◽

Pseudomonas Aeruginosa ◽

Enterococcus Faecalis ◽

Streptococcus Pyogenes ◽

Mentha Arvensis ◽

Ocimum Gratissimum ◽

Mueller Hinton ◽

Plectranthus Amboinicus

Objetivos: avaliar a atividade antimicrobiana em extrato aquoso, hidroalcoólico e alcoólico das folhas de espécies da família Lamiaceae frente a bactérias de interesse. Método: Foram escolhidas quatro espécies: Ocimum gratissimum, Plectranthus amboinicus, Mentha arvensis e Plectranthus barbatus. A partir das folhas foram confeccionados os extratos aquoso, hidroalcoólico e alcoólico nas concentrações 100mg/mL, 50mg/mL e 25mg/mL. Foram selecionadas as bactérias Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus e Pseudomonas aeruginosa para os ensaios de antibiose em Ágar Mueller-Hinton. Resultados: P. barbatus, em seu extrato hidroalcoólico mostrou ativo nas três concentrações para bactéria S. aureus, e ainda foi ativo para P. aeruginosa, demonstrando no extrato alcoólico atividade frente as bactérias. Para M. arvensis e P. amboinicus, seus extratos hidroalcoólico e alcoólico apresentaram atividade para S. aureus. Conclusão: Sugere-se que as espécies em questão apresentem boa atividade antimicrobiana, sendo necessária a realização de mais estudos para melhor entender esse mecanismo.

Download Full-text