Tools, strains, and strategies to effectively conduct anaerobic and aerobic transcriptional reporter screens and assays in Staphylococcus aureus

Transcriptional reporters are reliable and time-tested tools to study gene regulation. In Staphylococcus aureus, β-galactosidase (lacZ)-based genetic screens are not widely used because of the necessity of selectable markers for strain construction and the production of staphyloxanthin pigment which obfuscates results. We describe a series of vectors that allow for markerless insertion of codon-optimized lacZ-based transcriptional reporters. The vectors encode for different ribosomal binding sites allowing for tailored lacZ expression. A ΔcrtM::kanR deletion insertion mutant was constructed that prevents the synthesis of staphyloxanthin, thereby permitting blue-white screening without the interference of carotenoid production. We demonstrate the utility of these vectors to monitor aerobic and anaerobic transcriptional activity. For the latter, we describe the use of a ferrocyanide-ferricyanide redox system (Fe(CN)63–/4–) permitting blue-white screening in the absence of oxygen. We also describe additional reporter systems and methods for monitoring transcriptional activity during anaerobic culture including a FAD-binding fluorescent protein (EcFbFP), alpha-hemolysin (hla), or lipase (geh). The systems and methods described are compatible with vectors utilized to create and screen high-density transposon mutant libraries.

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Chromosomal map location of a determinant affecting [alpha]-hemolysin production in Staphylococcus aureus

10.31274/rtd-180813-258 ◽

1978 ◽

Author(s):

Daniel Raymond Brown

Keyword(s):

Staphylococcus Aureus ◽

Alpha Hemolysin ◽

Chromosomal Map ◽

Map Location

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Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Scientific Reports ◽

10.1038/s41598-021-84505-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Andi R. Sultan ◽

Kirby R. Lattwein ◽

Nicole A. Lemmens-den Toom ◽

Susan V. Snijders ◽

Klazina Kooiman ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Clonal Complex ◽

Regulatory Pathways ◽

Immune Modulator ◽

Viability Assay ◽

Analgesic Agents ◽

Green Fluorescent

AbstractStaphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

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Functional expression of the alpha-hemolysin of Staphylococcus aureus in intact Escherichia coli and in cell lysates. Deletion of five C-terminal amino acids selectively impairs hemolytic activity.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)50103-x ◽

1992 ◽

Vol 267 (15) ◽

pp. 10902-10909

Author(s):

B Walker ◽

M Krishnasastry ◽

L Zorn ◽

J Kasianowicz ◽

H Bayley

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Staphylococcus Aureus ◽

Hemolytic Activity ◽

Functional Expression ◽

Cell Lysates ◽

Alpha Hemolysin ◽

Terminal Amino

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Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

Molecular and Cellular Biology ◽

10.1128/mcb.22.11.3852-3863.2002 ◽

2002 ◽

Vol 22 (11) ◽

pp. 3852-3863 ◽

Cited By ~ 59

Author(s):

Li Liu ◽

Michael C. Rice ◽

Miya Drury ◽

Shuqiu Cheng ◽

Howard Gamper ◽

...

Keyword(s):

Transcriptional Activity ◽

Fluorescent Protein ◽

Fusion Gene ◽

Strand Bias ◽

Nucleotide Exchange ◽

Gene Repair ◽

Protein Fusion ◽

Template Strand ◽

Targeted Gene Repair ◽

Targeting Vector

ABSTRACT Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.

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Improved Protection in a Rabbit Model of Community-Associated Methicillin-Resistant Staphylococcus aureus Necrotizing Pneumonia upon Neutralization of Leukocidins in Addition to Alpha-Hemolysin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01213-16 ◽

2016 ◽

Vol 60 (10) ◽

pp. 6333-6340 ◽

Cited By ~ 37

Author(s):

Binh An Diep ◽

Vien T. M. Le ◽

Zehra C. Visram ◽

Harald Rouha ◽

Lukas Stulik ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Human Monoclonal Antibody ◽

Rabbit Model ◽

Passive Immunization ◽

Severe Pneumonia ◽

Phagocytic Cells ◽

Methicillin Resistant ◽

Content Type ◽

Alpha Hemolysin ◽

Full Protection

ABSTRACTCommunity-associated methicillin-resistantStaphylococcus aureus(CA-MRSA), especially the USA300 pulsotype, is a frequent cause of skin and soft tissue infections and severe pneumonia. Despite appropriate antibiotic treatment, complications are common and pneumonia is associated with high mortality.S. aureusstrains express multiple cytotoxins, including alpha-hemolysin (Hla) and up to five bicomponent leukocidins that specifically target phagocytic cells for lysis. CA-MRSA USA300 strains carry the genes for all six cytotoxins. Species specificity of the leukocidins greatly contributes to the ambiguity regarding their role inS. aureuspathogenesis. We performed a comparative analysis of the leukocidin susceptibility of human, rabbit, and mouse polymorphonuclear leukocytes (PMNs) to assess the translational value of mouse and rabbitS. aureusmodels. We found that mouse PMNs were largely resistant to LukSF-PV, HlgAB, and HlgCB and susceptible only to LukED, whereas rabbit and human PMNs were highly sensitive to all these cytotoxins. In the rabbit pneumonia model with a USA300 CA-MRSA strain, passive immunization with a previously identified human monoclonal antibody (MAb), Hla-F#5, which cross-neutralizes Hla, LukSF-PV, HlgAB, HlgCB, and LukED, provided full protection, whereas an Hla-specific MAb was only partially protective. In the mouse USA300 CA-MRSA pneumonia model, both types of antibodies demonstrated full protection, suggesting that Hla, but not leukocidin(s), is the principal virulence determinant in mice. As the rabbit recapitulates the high susceptibility to leukocidins characteristic of humans, this species represents a valuable model for assessing novel, cytotoxin-targeting anti-S. aureustherapeutic approaches.

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Role of Antibodies in Protection Elicited by Active Vaccination with Genetically Inactivated Alpha Hemolysin in a Mouse Model of Staphylococcus aureus Skin and Soft Tissue Infections

Clinical and Vaccine Immunology ◽

10.1128/cvi.00051-14 ◽

2014 ◽

Vol 21 (5) ◽

pp. 622-627 ◽

Cited By ~ 19

Author(s):

Christopher P. Mocca ◽

Rebecca A. Brady ◽

Drusilla L. Burns

Keyword(s):

Staphylococcus Aureus ◽

Soft Tissue ◽

Murine Model ◽

Bacterial Colonization ◽

Passive Immunization ◽

The United States ◽

Soft Tissue Infections ◽

Content Type ◽

Alpha Hemolysin ◽

Active Vaccination

ABSTRACTDue to the emergence of highly virulent community-associated methicillin-resistantStaphylococcus aureus(CA-MRSA) infections,S. aureushas become a major threat to public health. A majority of CA-MRSA skin and soft tissue infections in the United States are caused byS. aureusUSA300 strains that are known to produce high levels of alpha hemolysin (Hla). Therefore, vaccines that contain inactivated forms of this toxin are currently being developed. In this study, we sought to determine the immune mechanisms of protection for this antigen using a vaccine composed of a genetically inactivated form of Hla (HlaH35L). Using a murine model of skin and soft tissue infections (SSTI), we found that BALB/c mice were protected by vaccination with HlaH35L; however, Jh mice, which are deficient in mature B lymphocytes and lack IgM and IgG in their serum, were not protected. Passive immunization with anti-HlaH35L antibodies conferred protection against bacterial colonization. Moreover, we found a positive correlation between the total antibody concentration induced by active vaccination and reduced bacterial levels. Animals that developed detectable neutralizing antibody titers after active vaccination were significantly protected from infection. These data demonstrate that antibodies to Hla represent the major mechanism of protection afforded by active vaccination with inactivated Hla in this murine model of SSTI, and in this disease model, antibody levels correlate with protection. These results provide important information for the future development and evaluation ofS. aureusvaccines.

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Decreased Vancomycin Susceptibility in Staphylococcus aureus Caused by IS256Tempering of WalKR Expression

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00279-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3240-3249 ◽

Cited By ~ 34

Author(s):

Christopher R. E. McEvoy ◽

Brian Tsuji ◽

Wei Gao ◽

Torsten Seemann ◽

Jessica L. Porter ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Fluorescent Protein ◽

Site Directed Mutagenesis ◽

Infection Model ◽

Content Type ◽

Reverse Transcription Pcr ◽

Phenotype Analysis ◽

Dosing Regimens ◽

Mrsa Strains ◽

Green Fluorescent

ABSTRACTVancomycin-intermediateStaphylococcus aureus(VISA) strains often arise by mutations in the essential two-component regulatorwalKR; however their impact onwalKRfunction has not been definitively established. Here, we investigated 10 MRSA strains recovered serially after exposure of vancomycin-susceptibleS. aureus(VSSA) JKD6009 to simulated human vancomycin dosing regimens (500 mg to 4,000 mg every 12 h) using a 10-day hollow fiber infection model. After continued exposure to the vancomycin regimens, two isolates displayed reduced susceptibility to both vancomycin and daptomycin, developing independent IS256insertions in thewalKR5′ untranslated region (5′ UTR). Quantitative reverse transcription-PCR (RT-PCR) revealed a 50% reduction inwalKRgene expression in the IS256mutants compared to the VSSA parent. Green fluorescent protein (GFP) reporter analysis, promoter mapping, and site-directed mutagenesis confirmed these findings and showed that the IS256insertions had replaced two SigA-likewalKRpromoters with weaker, hybrid promoters. Removal of IS256reverted the phenotype to VSSA, showing that reduced expression of WalKR did induce the VISA phenotype. Analysis of selected WalKR-regulated autolysins revealed upregulation ofssaAbut no change in expression ofsakandsceDin both IS256mutants. Whole-genome sequencing of the two mutants revealed an additional IS256insertion withinagrCfor one mutant, and we confirmed that this mutation abolishedagrfunction. These data provide the first substantial analysis ofwalKRpromoter function and show that prolonged vancomycin exposure can result in VISA through an IS256-mediated reduction inwalKRexpression; however, the mechanisms by which this occurs remain to be determined.

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Membrane mutations and production of enterotoxin B and alpha hemolysin in Staphylococcus aureus

Canadian Journal of Microbiology ◽

10.1139/m75-039 ◽

1975 ◽

Vol 21 (3) ◽

pp. 275-280 ◽

Cited By ~ 3

Author(s):

Robert A. Altenbern

Keyword(s):

Staphylococcus Aureus ◽

Aureus Strain ◽

Type B ◽

Alpha Hemolysin ◽

Osmotic Stability ◽

Enterotoxin B

Staphylococcus aureus strain S-6, which produces enterotoxin type B (SEB), and strain 10-275, a high toxin-producing mutant derived from S-6, display pronounced differences in dye sensitivity, osmotic stability, and bacitracin sensitivity. Such characteristics are consistent with the concept that strain 10-275 is a membrane mutant of strain S-6. Some membrane mutants of S. aureus strain 14458 exhibit about two- to three-fold increases in SEB production whereas other membrane mutants show about twofold increases in α-hemolysin production. It is suggested that specific and independent membrane mutations control the secretory processes resulting in the extracellular elaboration of these exoproteins.

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Porcine OCT4 Reporter System can Monitor Species-Specific Pluripotency During Somatic Cell Reprogramming

10.21203/rs.3.rs-59439/v1 ◽

2020 ◽

Author(s):

Seung-Hun Kim ◽

Kwang-Hwan Choi ◽

Mingyun Lee ◽

Dong-Kyung Lee ◽

Chang-Kyu Lee

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Fluorescent Protein ◽

Upstream Region ◽

Reporter System ◽

System L ◽

Reporter Systems ◽

Induced Pluripotent ◽

Species Specific ◽

Fluorescent Protein Reporter

Abstract l Background: The present study examined the activity and function of pig OCT4 enhancer in porcine reprogramming cells. Dual fluorescent protein reporter systems controlled by the upstream regulatory region of OCT4, which is one of the master regulators for pluripotency, are widely used in studies of the mechanism of pluripotency. We analyzed how this reporter system functions in FGF- or LIF-dependent reprogrammed porcine pluripotent stem cells using the previously established porcine-specific reporter system. l Results: Porcine embryonic fibroblasts were coinfected with the pOCT4-∆PE-eGFP (DE-GFP) and pOCT4-∆DE-DsRed2 (PE-RFP) vectors, and GFP and RFP expression was verified during a DOX-dependent reprogramming process. We demonstrated that the porcine OCT4 distal enhancer and proximal enhancer were activated in different expression patterns simultaneously as the changes in the expression of pluripotent marker genes during the establishment of porcine-induced pluripotent stem cells (iPSCs). l Conclusions: Porcine OCT4 upstream region-derived dual fluorescent protein reporter systems serve as live naïve/primed pluripotency indicators for porcine induced pluripotent cell establishment. This work demonstrates the applicability of the porcine OCT4 upstream region-derived dual fluorescence reporter system, which may be applied to investigations of species-specific pluripotency in porcine-origin cells. These reporter systems may be useful tools for studies of porcine-specific pluripotency, early embryo development and embryonic stem cells.

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