scholarly journals The Intracellular Cyclophilin PpiB Contributes to the Virulence ofStaphylococcus aureusIndependently of Its Peptidyl-Prolylcis/transIsomerase Activity

2018 ◽  
Vol 86 (11) ◽  
Author(s):  
Rebecca A. Keogh ◽  
Rachel L. Zapf ◽  
Richard E. Wiemels ◽  
Marcus A. Wittekind ◽  
Ronan K. Carroll
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Content Type ◽  
Allelic Exchange ◽  
Isomerase Activity

ABSTRACTTheStaphylococcus aureuscyclophilin PpiB is an intracellular peptidyl prolylcis/transisomerase (PPIase) that has previously been shown to contribute to secreted nuclease and hemolytic activity. In this study, we investigated the contribution of PpiB toS. aureusvirulence. Using a murine abscess model of infection, we demonstrated that appiBmutant is attenuated for virulence. We went on to investigate the mechanism through which PpiB protein contributes to virulence, in particular the contribution of PpiB PPIase activity. We determined the amino acid residues that are important for PpiB PPIase activity and showed that a single amino acid substitution (F64A) completely abrogates PPIase activity. Using purified PpiB F64A proteinin vitro, we showed that PPIase activity only partially contributes to Nuc refolding and that PpiB also possesses PPIase-independent activity. Using allelic exchange, we introduced the F64A substitution onto theS. aureuschromosome, generating a strain that produces enzymatically inactive PpiB. Analysis of the PpiB F64A strain revealed that PPIase activity is not required for hemolysis of human blood or virulence in a mouse. Together, these results demonstrate that PpiB contributes toS. aureusvirulence via a mechanism unrelated to prolyl isomerase activity.

scholarly journals Susceptibility of Methicillin-Resistant Staphylococcus aureus to Five Quinazolinone Antibacterials

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Sara Ceballos ◽  
Choon Kim ◽  
Yuanyuan Qian ◽  
Shahriar Mobashery ◽  
Mayland Chang ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Binding Proteins ◽  
Methicillin Resistant Staphylococcus Aureus ◽  
Methicillin Resistant ◽  
Content Type ◽  
Penicillin Binding Proteins

ABSTRACT The in vitro activities of five quinazolinone antibacterials, compounds Q1 to Q5, were tested against 210 strains of methicillin-resistant Staphylococcus aureus (MRSA). The MIC50/MIC90 values (in μg/ml) were as follows: Q1, 0.5/2; Q2, 1/4; Q3, 2/4; Q4, 0.06/0.25; and Q5, 0.125/0.5. Several strains with high MIC values (from 8 to >32 μg/ml) for some of these compounds exhibited amino acid changes in the penicillin-binding proteins, which are targeted by these antibacterials.

scholarly journals Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal

2014 ◽  
Vol 13 (4) ◽  
pp. 539-547 ◽  
Author(s):  
VaNae Hamilton ◽  
Ujjal K. Singha ◽  
Joseph T. Smith ◽  
Ebony Weems ◽  
Minu Chaudhuri
Keyword(s):  
Amino Acid ◽  
Alternative Oxidase ◽  
Heterologous Protein ◽  
Amino Acid Residues ◽  
Mitochondrial Targeting ◽  
Content Type ◽  
Targeting Signal ◽  
Internal Signal ◽  
Trypanosome Alternative Oxidase

ABSTRACTRecognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form ofTrypanosoma brucei. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondriain vitro. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to aT. bruceimitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.

scholarly journals Reconstruction of the immunogenic peptide RNase(43-56) by identification and transfer of the critical residues into an unrelated peptide backbone.

1989 ◽  
Vol 170 (1) ◽  
pp. 203-215 ◽  
Author(s):  
R G Lorenz ◽  
A N Tyler ◽  
P M Allen
Keyword(s):  
Amino Acid ◽  
T Cell ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Peptide Backbone ◽  
Chimeric Peptide ◽  
Critical Residues

The involvement of each of the amino acid residues of the I-Ak-restricted T cell determinant RNase(43-56) was examined in detail using a series of peptides containing single amino acid substitutions. Four positions were identified as being essential for the formation of the determinant, Phe-46, Val-47, His-48, and Leu-51. When these four residues were substituted into the backbone of the unrelated peptide HA(130-144), a nonstimulatory peptide was obtained. The inclusion of an additional residue, Val-54, resulted in a chimeric peptide, RN/HA2, which was nearly as active as the native molecule. The peptide RN/HA2 was able to prime in vivo for RNase reactivity, confirming that these five residues contained all of the specificity of the RNase(43-56) determinant. The role of three of these critical residues was examined using both a functional competition assay and an in vivo priming assay. It was ascertained that the Phe-46 was directly involved in contacting the TCR, while the His-48 and Leu-51 were either involved in binding to the I-Ak molecule or in determining the conformation of the peptide. Thus, by critically evaluating the contribution of each of the amino acid residues in a T cell determinant, we were able to generate a chimeric peptide only containing 5 of 15 residues from the RNase(43-56) sequence that was functionally identical to the native RNase(43-56) molecule both in vitro and in vivo.

scholarly journals The Peptidyl-Prolyl Isomerase Activity of SlyD Is Not Required for Maturation of Escherichia coli Hydrogenase

2007 ◽  
Vol 189 (21) ◽  
pp. 7942-7944 ◽  
Author(s):  
Jie Wei Zhang ◽  
Michael R. Leach ◽  
Deborah B. Zamble
Keyword(s):  
Escherichia Coli ◽  
Metal Cluster ◽  
The Other ◽  
Prolyl Isomerase ◽  
Ppiase Activity ◽  
Peptidyl Prolyl Isomerase ◽  
Isomerase Activity ◽  
Hydrogenase Enzymes

ABSTRACT Escherichia coli SlyD, which is involved in the biosynthesis of the metal cluster in the [NiFe]-hydrogenase enzymes, exhibits several activities including that of a peptidyl-prolyl isomerase (PPIase). Mutations that result in deficient PPIase activity do not produce corresponding decreases in the other activities of SlyD in vitro or in hydrogenase production levels in vivo.

scholarly journals An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

2016 ◽  
Vol 199 (1) ◽  
Author(s):  
Richard E. Wiemels ◽  
Stephanie M. Cech ◽  
Nikki M. Meyer ◽  
Caleb A. Burke ◽  
Andy Weiss ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Active Form ◽  
Ppiase Activity ◽  
Content Type ◽  
Associated Proteins

ABSTRACT Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus. IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

scholarly journals Candida albicans Induces Arginine Biosynthetic Genes in Response to Host-Derived Reactive Oxygen Species

2012 ◽  
Vol 12 (1) ◽  
pp. 91-100 ◽  
Author(s):  
Claudia Jiménez-López ◽  
John R. Collette ◽  
Kimberly M. Brothers ◽  
Kelly M. Shepardson ◽  
Robert A. Cramer ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Candida Albicans ◽  
Amino Acid ◽  
Reactive Oxygen ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Oxygen Species ◽  
Biosynthetic Genes ◽  
Content Type

ABSTRACTThe interaction ofCandida albicanswith phagocytes of the host's innate immune system is highly dynamic, and its outcome directly impacts the progression of infection. While the switch to hyphal growth within the macrophage is the most obvious physiological response, much of the genetic response reflects nutrient starvation: translational repression and induction of alternative carbon metabolism. Changes in amino acid metabolism are not seen, with the striking exception of arginine biosynthesis, which is upregulated in its entirety during coculture with macrophages. Using single-cell reporters, we showed here that arginine biosynthetic genes are induced specifically in phagocytosed cells. This induction is lower in magnitude than during arginine starvationin vitroand is driven not by an arginine deficiency within the phagocyte but instead by exposure to reactive oxygen species (ROS). Curiously, these genes are induced in a narrow window of sublethal ROS concentrations.C. albicanscells phagocytosed by primary macrophages deficient in thegp91phoxsubunit of the phagocyte oxidase do not express theARGpathway, indicating that the induction is dependent on the phagocyte oxidative burst.C. albicans argpathway mutants are retarded in germ tube and hypha formation within macrophages but are not notably more sensitive to ROS. We also find that theARGpathway is regulated not by the general amino acid control response but by transcriptional regulators similar to theSaccharomyces cerevisiaeArgR complex. In summary, phagocytosis induces this single amino acid biosynthetic pathway in an ROS-dependent manner.

Generating Cyan Fluorescence with De Novo Tripeptides: An In Vitro Mutation Study on the Role of Single Amino Acid Residues and Their Sequence

ChemBioChem ◽  
2019 ◽  
Vol 20 (18) ◽  
pp. 2324-2330 ◽  
Author(s):  
Jun Guo ◽  
Srinivasan Ramachandran ◽  
Ruibo Zhong ◽  
Ratnesh Lal ◽  
Feng Zhang
Keyword(s):  
Amino Acid ◽  
De Novo ◽  
Single Amino Acid ◽  
Amino Acid Residues

scholarly journals Protective Role of d-Amino Acid Oxidase against Staphylococcus aureus Infection

2012 ◽  
Vol 80 (4) ◽  
pp. 1546-1553 ◽  
Author(s):  
Hideaki Nakamura ◽  
Jun Fang ◽  
Hiroshi Maeda
Keyword(s):  
Hydrogen Peroxide ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Hypochlorous Acid ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Content Type

ABSTRACTd-Amino acid oxidase (DAO) is a hydrogen peroxide-generating enzyme that uses ad-amino acid as a substrate. We hypothesized that DAO may protect against bacterial infection, because hydrogen peroxide is one of the most important molecules in the antibacterial defense systems in mammals. We show here that DAO suppressed the growth ofStaphylococcus aureusin a manner that depended on the concentration of DAO andd-amino acidin vitro. Addition of catalase abolished the bacteriostatic activity of DAO. Although DAO plusd-Ala showed less bactericidal activity, addition of myeloperoxidase (MPO) greatly enhanced the bactericidal activity of DAO. Furthermore, DAO was able to utilize bacterial lysate, which containsd-Ala derived from peptidoglycan; this could produce hydrogen peroxide with, in the presence of myeloperoxidase, formation of hypochlorous acid. This concerted reaction of DAO and MPO led to the bactericidal action.In vivoexperiments showed that DAO−/−(mutant) mice were more susceptible toS. aureusinfection than were DAO+/+(wild-type) mice. These results suggest that DAO, together with myeloperoxidase, may play an important role in antibacterial systems in mammals.

scholarly journals Antifungal Susceptibility Profiles and Drug Resistance Mechanisms of Clinical Lomentospora prolificans Isolates

2020 ◽  
Vol 64 (11) ◽  
Author(s):  
Yongqin Wu ◽  
Nina Grossman ◽  
Marissa Totten ◽  
Warda Memon ◽  
Anna Fitzgerald ◽  
...  
Keyword(s):  
Amino Acid ◽  
Tandem Repeats ◽  
Hot Spot ◽  
Antifungal Susceptibility ◽  
Antifungal Drugs ◽  
Upstream Region ◽  
Amino Acid Residues ◽  
Content Type ◽  
Echinocandin Resistance

ABSTRACT Lomentospora prolificans is an opportunistic fungal pathogen with low susceptibility to current antifungal drugs. Here, we tested the in vitro susceptibility of 8 drugs against 42 clinical L. prolificans isolates. All isolates showed high MICs to voriconazole (MIC90>16 μg/ml), itraconazole (MIC90>16 μg/ml), posaconazole (MIC90>16 μg/ml), isavuconazole (MIC90>16 μg/ml), amphotericin B (MIC90>16 μg/ml), and terbinafine (MIC90>64 μg/ml) and high minimum effective concentrations (MECs) to micafungin (MEC90>8 μg/ml), with the exception of miltefosine showing an MIC90 value of 4 μg/ml. We examined six different in vitro drug combinations and found that the combination of voriconazole and terbinafine achieved the most synergistic effort against L. prolificans. We then annotated the L. prolificans whole genome and located its Cyp51 and Fks1 genes. We completely sequenced the two genes to determine if any mutation would be related to azole and echinocandin resistance in L. prolificans. We found no amino acid changes in Cyp51 protein and no tandem repeats in the 5′ upstream region of the Cyp51 gene. However, we identified three intrinsic amino acid residues (G138S, M220I, and T289A) in the Cyp51 protein that were linked to azole resistance. Likewise, two intrinsic amino acid residues (F639Y, W695F) that have reported to confer echinocandin resistance were found in Fks1 hot spot regions. In addition, three new amino acid alterations (D440A, S634R, and H1245R) were found outside Fks1 hot spot regions, and their contributions to echinocandin resistance need future investigation. Overall, our findings support the notion that L. prolificans is intrinsically resistant to azoles and echinocandins.

scholarly journals Amino Acid Substitutions Account for Most MexS Alterations in ClinicalnfxCMutants of Pseudomonas aeruginosa

2016 ◽  
Vol 60 (4) ◽  
pp. 2302-2310 ◽  
Author(s):  
Charlotte Richardot ◽  
Paulo Juarez ◽  
Katy Jeannot ◽  
Isabelle Patry ◽  
Patrick Plésiat ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Hospital Setting ◽  
Multidrug Resistant ◽  
Single Amino Acid ◽  
Content Type ◽  
Active Efflux ◽  
Amino Acid Mutations ◽  
Virulence Factor Production

ABSTRACTMultidrug-resistant mutants ofPseudomonas aeruginosathat overproduce the active efflux system MexEF-OprN (callednfxCmutants) have rarely been characterized in the hospital setting. Screening of 221 clinical strains exhibiting a reduced susceptibility to ciprofloxacin (a substrate of MexEF-OprN) and imipenem (a substrate of the negatively coregulated porin OprD) led to the identification of 43 (19.5%)nfxCmutants. Subsequent analysis of 22 nonredundant mutants showed that, in contrast to theirin vitro-selected counterparts, only 3 of them (13.6%) harbored a disruptedmexSgene, which codes for the oxidoreductase MexS, whose inactivation is known to activate themexEF-oprNoperon through a LysR-type regulator, MexT. Nine (40.9%) of the clinicalnfxCmutants contained single amino acid mutations in MexS, and these were associated with moderate effects on resistance and virulence factor production in 8/9 strains. Finally, the remaining 10 (45.5%)nfxCmutants did not display mutations in any of the regulators known to controlmexEF-oprNexpression (themexS,mexT,mvaT, andampRgenes), confirming that other loci are responsible for pump upregulation in patients. Collectively, these data demonstrate thatnfxCmutants are probably more frequent in the hospital than previously thought and have genetic and phenotypic features somewhat different from those ofin vitro-selected mutants.

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