Genetic Loci of Streptococcus mitis That Mediate Binding to Human Platelets

ABSTRACT The direct binding of bacteria to platelets is a postulated major interaction in the pathogenesis of infective endocarditis. To identify bacterial components that mediate platelet binding byStreptococcus mitis, we screened a Tn916ΔE-derived mutant library of S. mitisstrain SF100 for reduced binding to human platelets in vitro. Two distinct loci were found to affect platelet binding. The first contains a gene (pblT) encoding a highly hydrophobic, 43-kDa protein with 12 potential membrane-spanning segments. This protein resembles members of the major facilitator superfamily of small-molecule transporters. The second platelet binding locus consists of an apparent polycistronic operon. This region includes genes that are highly similar to those of Lactococcus lactis phage r1t andStreptococcus thermophilus phage 01205. Two genes (pblA and pblB) encoding large surface proteins are also present. The former encodes a 107-kDa protein containing tryptophan-rich repeats, which may serve to anchor the protein within the cell wall. The latter encodes a 121-kDa protein most similar to a tail fiber protein from phage 01205. Functional mapping by insertion-duplication mutagenesis and gene complementation indicates that PblB may be a platelet adhesin and that expression of PblB may be linked to that of PblA. The combined data indicate that at least two genomic regions contribute to platelet binding by S. mitis.One encodes a probable transmembrane transporter, while the second encodes two large surface proteins resembling structural components of lysogenic phages.

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Adaptive Laboratory Evolution for Multistress Tolerance, including Fermentability at High Glucose Concentrations in Thermotolerant Candida tropicalis

Energies ◽

10.3390/en15020561 ◽

2022 ◽

Vol 15 (2) ◽

pp. 561

Author(s):

Koudkeo Phommachan ◽

Chansom Keo-oudone ◽

Mochamad Nurcholis ◽

Nookhao Vongvilaisak ◽

Mingkhuan Chanhming ◽

...

Keyword(s):

High Temperatures ◽

Candida Tropicalis ◽

High Glucose ◽

Glucose Transporter ◽

Major Facilitator Superfamily ◽

Cellulosic Biomass ◽

High Concentration ◽

Adaptive Laboratory Evolution ◽

Membrane Spanning ◽

Major Facilitator

Candida tropicalis, a xylose-fermenting yeast, has the potential for converting cellulosic biomass to ethanol. Thermotolerant C. tropicalis X-17, which was isolated in Laos, was subjected to repetitive long-term cultivation with a gradual increase in temperature (RLCGT) in the presence of a high concentration of glucose, which exposed cells to various stresses in addition to the high concentration of glucose and high temperatures. The resultant adapted strain demonstrated increased tolerance to ethanol, furfural and hydroxymethylfurfural at high temperatures and displayed improvement in fermentation ability at high glucose concentrations and xylose-fermenting ability. Transcriptome analysis revealed the up-regulation of a gene for a glucose transporter of the major facilitator superfamily and genes for stress response and cell wall proteins. Additionally, hydropathy analysis revealed that three genes for putative membrane proteins with multiple membrane-spanning segments were also up-regulated. From these findings, it can be inferred that the up-regulation of genes, including the gene for a glucose transporter, is responsible for the phenotype of the adaptive strain. This study revealed part of the mechanisms of fermentability at high glucose concentrations in C. tropicalis and the results of this study suggest that RLCGT is an effective procedure for improving multistress tolerance.

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Antimicrobial Activity of a Library of Thioxanthones and Their Potential as Efflux Pump Inhibitors

Pharmaceuticals ◽

10.3390/ph14060572 ◽

2021 ◽

Vol 14 (6) ◽

pp. 572

Author(s):

Fernando Durães ◽

Andreia Palmeira ◽

Bárbara Cruz ◽

Joana Freitas-Silva ◽

Nikoletta Szemerédi ◽

...

Keyword(s):

Quorum Sensing ◽

Mammalian Cells ◽

Efflux Pump ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Design And Synthesis ◽

Resistance Nodulation Division ◽

Efflux Pump Inhibitors ◽

Major Facilitator

The overexpression of efflux pumps is one of the causes of multidrug resistance, which leads to the inefficacy of drugs. This plays a pivotal role in antimicrobial resistance, and the most notable pumps are the AcrAB-TolC system (AcrB belongs to the resistance-nodulation-division family) and the NorA, from the major facilitator superfamily. In bacteria, these structures can also favor virulence and adaptation mechanisms, such as quorum-sensing and the formation of biofilm. In this study, the design and synthesis of a library of thioxanthones as potential efflux pump inhibitors are described. The thioxanthone derivatives were investigated for their antibacterial activity and inhibition of efflux pumps, biofilm formation, and quorum-sensing. The compounds were also studied for their potential to interact with P-glycoprotein (P-gp, ABCB1), an efflux pump present in mammalian cells, and for their cytotoxicity in both mouse fibroblasts and human Caco-2 cells. The results concerning the real-time ethidium bromide accumulation may suggest a potential bacterial efflux pump inhibition, which has not yet been reported for thioxanthones. Moreover, in vitro studies in human cells demonstrated a lack of cytotoxicity for concentrations up to 20 µM in Caco-2 cells, with some derivatives also showing potential for P-gp modulation.

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O-acetylation controls the glycosylation of bacterial serine-rich repeat glycoproteins.

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016116 ◽

2020 ◽

pp. jbc.RA120.016116

Author(s):

Ravin Seepersaud ◽

Alexander C. Anderson ◽

Barbara A. Bensing ◽

Biswa P Choudhury ◽

Anthony J. Clarke ◽

...

Keyword(s):

Linkage Analysis ◽

Regulatory Mechanism ◽

Human Platelets ◽

Bacterial Virulence ◽

Wild Type ◽

Post Translational Modification ◽

Gram Positive Bacteria ◽

Wide Range ◽

Platelet Binding

The serine-rich repeat (SRR) glycoproteins of Gram-positive bacteria are a family of adhesins that bind to a wide range of host ligands, and expression of SRR glycoproteins is linked with enhanced bacterial virulence. The biogenesis of these surface glycoproteins involves their intracellular glycosylation and export via the accessory Sec (aSec) system. While all aSec components are required for SRR glycoprotein export, Asp2 of Streptococcus gordonii also functions as an O-acetyltransferase that modifies GlcNAc residues on the SRR adhesin GspB. Since these GlcNAc residues can also be modified by the glycosyltransferases Nss and Gly, it has been unclear whether the post-translational modification of GspB is coordinated. We now report that acetylation modulates the glycosylation of exported GspB. Loss of O-acetylation due to aps2 mutagenesis led to the export of GspB glycoforms with increased glucosylation of the GlcNAc moieties. Linkage analysis of the GspB glycan revealed that both O-acetylation and glucosylation occurred at the same C6 position on GlcNAc residues, and that O-acetylation prevented Glc deposition. Whereas streptococci expressing non-acetylated GspB with increased glucosylation were significantly reduced in their ability to bind human platelets in vitro, deletion of the glycosyltransferases nss and gly in the asp2 mutant restored platelet binding to wild-type levels. These findings demonstrate that GlcNAc O-acetylation controls GspB glycosylation, such that binding via this adhesin is optimized. Moreover, since O-acetylation has comparable effects on the glycosylation of other SRR adhesins, acetylation may represent a conserved regulatory mechanism for the post-translational modification of the SRR glycoprotein family.

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Molecular mechanisms associated with the resistance of Rhizoctonia solani AG-4 isolates to the succinate dehydrogenase inhibitor, thifluzamide

Phytopathology ◽

10.1094/phyto-06-21-0266-r ◽

2021 ◽

Author(s):

Can Zhao ◽

Yuting Li ◽

Zhijian Liang ◽

Lihong Gao ◽

Chenggui Han ◽

...

Keyword(s):

Succinate Dehydrogenase ◽

Molecular Mechanisms ◽

Major Facilitator Superfamily ◽

Cross Resistance ◽

Active Efflux ◽

Fitness Evaluation ◽

Reverse Transcription Pcr ◽

Major Facilitator ◽

Median Effective Concentration

Thifluzamide, a succinate dehydrogenase (SDH) inhibitor, possesses high activity against Rhizoctonia. In this study, 144 R. solani AG-4 (4HGI, 4HGII, and 4HGIII) isolates, the predominate pathogen associated with sugar beet seedling damping-off, were demonstrated to be sensitive to thifluzamide with a calculated mean median effective concentration of 0.0682 ± 0.0025 μg/mL. Thifluzamide-resistant isolates were generated using fungicide-amended media, resulting in four AG-4HGI isolates and eight AG-4HGII isolates with stable resistance and almost no loss in fitness. Evaluation of cross-resistance of the twelve thifluzamide-resistant isolates and their corresponding parental-sensitive isolates revealed a moderately positive correlation between thifluzamide resistance and the level of resistance to eight other fungicides from three groups, the exception being fludioxonil. An active efflux of fungicide through ATP-binding cassette and major facilitator superfamily transporters was found to be correlated to the resistance of R. solani AG-4HGII isolates to thifluzamide based on RNA-sequencing and quantitative reverse transcription-PCR analyses. Sequence analysis of sdhA, sdhB, sdhC, and sdhD revealed replacement of isoleucine by phenylalanine at position 61 in SDHC in nine of the twelve generated thifluzamide-resistant isolates. No other mutations were found in any of the other genes. Collectively, the data indicate that the active efflux of fungicide and a point mutation in sdhC may contribute to the resistance of R. solani AG-4HGI and AG-4HGII isolates to thifluzamide in vitro. This is the first characterization of the potential molecular mechanism associated with the resistance of R. solani AG-4 isolates to thifluzamide, and provides practical guidance for the use of this fungicide.

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Proteins PblA and PblB of Streptococcus mitis, Which Promote Binding to Human Platelets, Are Encoded within a Lysogenic Bacteriophage

Infection and Immunity ◽

10.1128/iai.69.10.6186-6192.2001 ◽

2001 ◽

Vol 69 (10) ◽

pp. 6186-6192 ◽

Cited By ~ 73

Author(s):

Barbara A. Bensing ◽

Ian R. Siboo ◽

Paul M. Sullam

Keyword(s):

Western Blot ◽

Mitomycin C ◽

Blot Analysis ◽

Western Blot Analysis ◽

Uv Light ◽

Human Platelets ◽

Surface Proteins ◽

Lytic Cycle ◽

Virulence Determinants ◽

Streptococcus Mitis

ABSTRACT The binding of platelets by bacteria is a proposed central mechanism in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 (an endocarditis isolate) was recently shown to be mediated in part by the surface proteins PblA and PblB. The genes encoding PblA and PblB are clustered with genes nearly identical to those of streptococcal phages r1t, 01205, and Dp-1, suggesting that pblA andpblB might reside within a prophage. To address this possibility, cultures of SF100 were exposed to either mitomycin C or UV light, both of which are known to induce the lytic cycle of many temperate phages. Both treatments caused a significant increase in the transcription of pblA. Treatment with mitomycin C or UV light also caused a substantial increase in the expression of PblA and PblB, as detected by Western blot analysis of proteins in the SF100 cell wall. By electron microscopy, phage particles were readily visible in the supernatants from induced cultures of SF100. The phage, designated SM1, had a double-stranded DNA genome of approximately 35 kb. Southern blot analysis of phage DNA indicated thatpblA and pblB were contained within the SM1 genome. Furthermore, Western blot analysis of phage proteins revealed that both PblA and PblB were present in the phage particles. These findings indicate that PblA and PblB are encoded by a lysogenic bacteriophage, which could facilitate the dissemination of these potential virulence determinants to other bacterial pathogens.

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Role of SraP, a Serine-Rich Surface Protein of Staphylococcus aureus, in Binding to Human Platelets

Infection and Immunity ◽

10.1128/iai.73.4.2273-2280.2005 ◽

2005 ◽

Vol 73 (4) ◽

pp. 2273-2280 ◽

Cited By ~ 156

Author(s):

Ian R. Siboo ◽

Henry F. Chambers ◽

Paul M. Sullam

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Molecular Mass ◽

Signal Sequence ◽

Rabbit Model ◽

Human Platelets ◽

Ligand Interaction ◽

Calculated Molecular Mass ◽

Platelet Binding

ABSTRACT The binding of bacteria to platelets is a postulated central event in the pathogenesis of infective endocarditis. Platelet binding by Streptococcus gordonii is mediated in large part by GspB, a high-molecular-mass cell wall glycoprotein. Although Staphylococcus aureus has a GspB homolog (SraP), little is known about its function. SraP has a calculated molecular mass of 227 kDa and, like GspB, is predicted to contain an atypical N-terminal signal sequence, two serine-rich repeat regions (srr1 and srr2) separated by a nonrepeat region, and a C-terminal cell wall anchoring motif (LPDTG). To assess whether SraP contributes to platelet binding, we compared the binding to human platelets of S. aureus strain ISP479C and of an isogenic variant (strain PS767) in which sraP had been disrupted by allelic replacement. Platelet binding in vitro by PS767 was 47% ± 17% (mean ± standard deviation) lower than that of ISP479C (P < 0.001). In addition, a recombinant fragment of SraP containing srr1 and the nonrepeat region was found to bind platelets directly. Binding was saturable, suggesting a receptor-ligand interaction. When tested in a rabbit model of endocarditis, in which each animal was simultaneously infected with ISP479C and PS767 at a ratio of approximately 1:1, the titers of the mutant strain within vegetations were significantly lower than those of the parent strain at 1 and 24 h postinfection. These results indicate that SraP can mediate the direct binding of S. aureus to platelets and that the platelet-binding domain of this glycoprotein is located within its N-terminal region. Moreover, the expression of SraP appears to be a virulence determinant in endovascular infection.

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Membrane transporter proteins are involved in Trichophyton rubrum pathogenesis

Journal of Medical Microbiology ◽

10.1099/jmm.0.002907-0 ◽

2009 ◽

Vol 58 (2) ◽

pp. 163-168 ◽

Cited By ~ 23

Author(s):

Fernanda C. A. Maranhão ◽

Fernanda G. Paião ◽

Ana Lúcia Fachin ◽

Nilce M. Martinez-Rossi

Keyword(s):

Abc Transporter ◽

Trichophyton Rubrum ◽

Disease Model ◽

Initial Step ◽

Functional Expression ◽

Infection Process ◽

Major Facilitator Superfamily ◽

Gene Encoding ◽

Major Facilitator

Trichophyton rubrum is a dermatophyte responsible for the majority of human superficial mycoses. The functional expression of proteins important for the initial step and the maintenance of the infection process were identified previously in T. rubrum by subtraction suppression hybridization after growth in the presence of keratin. In this study, sequences similar to genes encoding the multidrug-resistance ATP-binding cassette (ABC) transporter, copper ATPase, the major facilitator superfamily and a permease were isolated, and used in Northern blots to monitor the expression of the genes, which were upregulated in the presence of keratin. A sequence identical to the TruMDR2 gene, encoding an ABC transporter in T. rubrum, was isolated in these experiments, and examination of a T. rubrum ΔTruMDR2 mutant showed a reduction in infecting activity, characterized by low growth on human nails compared with the wild-type strain. The high expression levels of transporter genes by T. rubrum in mimetic infection and the reduction in virulence of the ΔTruMDR2 mutant in a disease model in vitro suggest that transporters are involved in T. rubrum pathogenicity.

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Streptococcus mitis Phage-Encoded Adhesins Mediate Attachment to α2-8-Linked Sialic Acid Residues on Platelet Membrane Gangliosides

Infection and Immunity ◽

10.1128/iai.01573-08 ◽

2009 ◽

Vol 77 (8) ◽

pp. 3485-3490 ◽

Cited By ~ 29

Author(s):

Jennifer Mitchell ◽

Paul M. Sullam

Keyword(s):

Human Platelets ◽

Sialic Acids ◽

Platelet Membrane ◽

Bacterial Cells ◽

Streptococcus Mitis ◽

Bacterial Binding ◽

Platelet Binding ◽

Platelet Membrane Receptor ◽

Direct Binding ◽

Ganglioside Gd3

ABSTRACT The direct binding of bacteria to human platelets contributes to the pathogenesis of infective endocarditis. Platelet binding by Streptococcus mitis strain SF100 is mediated in part by two bacteriophage-encoded proteins, PblA and PblB. However, the platelet membrane receptor for these adhesins has been unknown. In this study, we demonstrate that these proteins mediate attachment of bacterial cells to sialylated gangliosides on the platelet cell surface. Desialylation of human platelet monolayers reduced adherence of SF100, whereas treatment of the platelets with N- or O-glycanases did not affect platelet binding. Treatment of platelets with sialidases having different linkage specificities showed that removal of α2-8-linked sialic acids resulted in a marked reduction in bacterial binding. Preincubation of SF100 with ganglioside GD3, a glycolipid containing α2-8-linked sialic acids that is present on platelet membranes, blocked subsequent binding of this strain to these cells. In contrast, GD3 had no effect on the residual binding of platelets by strain PS344, an isogenic ΔpblA ΔpblB mutant. Preincubating platelets with specific monoclonal antibodies to ganglioside GD3 also inhibited binding of SF100 to platelets, but again, they had no effect on binding by PS344. When the direct binding of S. mitis strains SF100 and PS344 to immobilized gangliosides was tested, binding of PS344 to GD3 was reduced by 70% compared to the parent strain. These results indicated that platelet binding by SF100 is mediated by the interaction of PblA and PblB with α2-8-linked sialic acids on ganglioside GD3.

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Clumping Factor A Mediates Binding ofStaphylococcus aureus to Human Platelets

Infection and Immunity ◽

10.1128/iai.69.5.3120-3127.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3120-3127 ◽

Cited By ~ 81

Author(s):

Ian R. Siboo ◽

Ambrose L. Cheung ◽

Arnold S. Bayer ◽

Paul M. Sullam

Keyword(s):

Southern Blotting ◽

Human Platelets ◽

Platelet Membrane ◽

Start Codon ◽

Pcr Analysis ◽

Systemic Embolization ◽

Platelet Binding ◽

Platelet Membrane Receptor ◽

Direct Binding

ABSTRACT The direct binding of bacteria to platelets may be an important virulence mechanism in the pathogenesis of infective endocarditis. We have previously described Staphylococcus aureus strain PS12, a Tn551-derived mutant of strain ISP479, with reduced ability to bind human platelets in vitro. When tested in an animal model of endocarditis, the PS12 strain was less virulent than its parental strain, as measured by bacterial densities in endocardial vegetations and incidence of systemic embolization. We have now characterized the gene disrupted in PS12 and its function in platelet binding. DNA sequencing, Southern blotting, and PCR analysis indicate that PS12 contained two Tn551 insertions within the clumping factor A (ClfA) locus (clfA). The first copy was upstream from the clfA start codon and appeared to have no effect on ClfA production. The second insertion was within the region encoding the serine aspartate repeat of ClfA and resulted in the production of a truncated ClfA protein that was secreted from the cell. A purified, recombinant form of the ClfA A region, encompassing amino acids 40 through 559, significantly reduced the binding of ISP479C to human platelets by 44% (P = 0.0001). Immunoprecipitation of recombinant ClfA that had been incubated with solubilized platelet membranes coprecipitated a 118-kDa platelet membrane protein. This protein does not appear to be glycoprotein IIb. These results indicate that platelet binding by S. aureus is mediated in part by the direct binding of ClfA to a novel 118-kDa platelet membrane receptor.

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Evidence for a trap-and-flip mechanism in a proton-dependent lipid transporter

10.1101/2021.08.05.453402 ◽

2021 ◽

Author(s):

Elisabeth Lambert ◽

Ahmad Reza Mehdipour ◽

Alex Schmidt ◽

Gerhard Hummer ◽

Camilo Perez

Keyword(s):

Lipid Binding ◽

Major Facilitator Superfamily ◽

Common Mechanism ◽

Lipid Transporters ◽

Dynamics Simulations ◽

Major Facilitator ◽

Lipid Transporter ◽

Lipid Translocation

Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a trap-and-flip mechanism that might be shared among MFS lipid transporters.

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