Study of
            <i>Staphylococcus aureus</i>
            Pathogenic Genes by Transfer and Expression in the Less Virulent Organism
            <i>Streptococcus gordonii</i>

Meier, Patricia Stutzmann; Entenza, José M.; Vaudaux, Pierre; Francioli, P.; Glauser, M. P.; Moreillon, Philippe

doi:10.1128/iai.69.2.657-664.2001

Cited by 42 publications

(18 citation statements)

References 42 publications

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“…However, this difference was only recorded for a limited range of bacterial loads (33). In a reverse experiment, the S. aureus clfA gene was transferred into a surrogate, Streptococcus gordonii, which produced an increase in both bacterial adherence to platelet-fibrin clots and experimental endocarditis (46). As in the S. aureus knockout mutant, the difference between the parent and the transformant organisms was confined to an inoculum window of less than 1 order of magnitude.…”

Section: Discussionmentioning

confidence: 99%

“…Twenty-four hours after catheterization, groups of 10 rats were inoculated through a tail vein with increasing numbers of bacteria prepared from overnight cultures and sacrificed 24 h later. The rate and severity of valve infection were determined as described previously (33,46). The minimal inoculum infecting 80% of animals (80% infective dose [ID 80 ]) was assessed 24 h after bacterial challenge.…”

Section: Methodsmentioning

confidence: 99%

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Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

et al. 2001

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Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in singlegene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, both clfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as the S. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in >80% of the rats (80% infective dose [ID 80 ]) with the parent lactococcus was >10 7 CFU. In contrast, clfA-expressing and fnbA-expressing lactococci required only 10 5 CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID 80 ‫؍‬ 10 4 to 10 5 CFU) in this model. The results confirmed the role of clfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of the clfA and fnbA products should be blocked for the therapy to be effective.Staphylococcus aureus is a major pathogen responsible for a wide variety of infections. These range from relatively mild skin and soft-tissue diseases to severe conditions such as osteomyelitis and endocarditis (35,52). S. aureus also often infects foreign materials (11,19,48,49), thus making it a primary challenge for modern medicine. Finally, S. aureus has successfully collected numerous antibiotic resistance genes and can withstand treatment with the majority of nonexperimental antibacterial drugs, including now glycopeptides (20,40,45). This increases the spectrum of untreatable infections and warrants the search for alternative antistaphylococcal strategies.Understanding the steps of colonization and invasion of the host by the microorganism might greatly help define new targets to interfere with the infective process. However, identifying the key molecular players mediating S. aureus colonization and invasion has been hampered by the multiplicity of pathogenic factors expressed by the organisms. Indeed, S. aureus carries several functionally redundant adhesins binding to both soluble and insoluble components of the host extracellular matrix (39,53). Moreover, the analysis is complicated by the fact that expression of these determinants is differentially regulated by global regulators agr and sar (3, 38), which promote adhesin expression ea...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

et al. 2001

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“…However, transposon mutagenesis of emb, the S. defectivus homologue of ebh, abolished the ECM adherence of that organism (33). The functions of single proteins can be elucidated by cloning them into nonpathgenic bacteria such as Lactococcus lactis (44) or Streptococcus gordonii (49). In the case of Ebh it would not be technically feasible to express the whole protein given its very large size, but it may be possible to express a section of it, such as H2, although there would also be a requirement for artificial attachment to the cell surface, which could alter the behavior of the peptide.…”

Section: Discussionmentioning

confidence: 99%

Analysis of Ebh, a 1.1-Megadalton Cell Wall-Associated Fibronectin-Binding Protein of Staphylococcus aureus

et al. 2002

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In order for Staphylococcus aureus to adhere to host extracellular matrix (ECM) substrates, it elicits a wide range of surface proteins. We have characterized a novel ϳ1.1-MDa protein in S. aureus, termed Ebh (for ECMbinding protein homologue), which has homology to other ECM-binding proteins. Ebh consists of several domains, including a large central region with 44 imperfect repeats of 126 amino acids. Expression analysis revealed ebh to be growth phase regulated and repressed by agr. A fragment of the central repeat region of Ebh was cloned, overexpressed, and used in ligand-binding studies to determine Ebh function. The recombinant protein was found to specifically bind human fibronectin. Ebh is produced during human infection since serum samples taken from patients with confirmed S. aureus infections were found to contain anti-Ebh antibodies. Localization studies revealed Ebh to be cell envelope associated and is proposed to form a specialized surface structure involved in cellular adhesion.Staphylococcus aureus is able to cause a wide range of different infections, such as endocarditis, arthritis, and septicemia (55). In order for S. aureus to colonize and disseminate through its host, the bacterium expresses an array of proteins which interact with molecules of the host extracellular matrix (ECM). These bacterial cell surface and extracellular proteins bind to a wide range of host proteins, such as fibronectin (Fn) (15,21,25,42), fibrinogen (Fg) (21,36,40,56), vitronectin (21), collagen (50), thrombospondin (18), bone sialoprotein (51), elastin (8), and von Willebrand factor (16), belying the ability of S. aureus to act as the etiological agent of a variety of pathologies. Fn is an adhesive glycoprotein found on the surface of mammalian cells and in serum (7). Previous studies have implicated staphylococcal Fn-binding proteins with adhesion to different cell types (1,23,37,38,47). Also, they bind Fn, which acts as an invasin, forming a bridge between S. aureus and an integrin on the surface of nonprofessional phagocytes (1,9,23,33,46,48). Most of the ligand-binding proteins that have previously been characterized are found associated with the cell wall and are known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (13). The specific interactions that these adhesins undergo with the ECM, coupled with the fact that they are found on the surface of the cell, means that they may be useful targets for prophylaxis or therapy, e.g., as vaccine components, as targets for passive immunotherapy, or for novel antiadhesive strategies.Recently, the genomes of S. aureus strains N315 and Mu50 have been published (31), and determination of the genomes of five additional strains has either been done or is nearing completion. These studies have revealed the presence of many uncharacterized putative surface proteins. The two largest genes, ebhA and ebhB, encode putative proteins of ca. 722 and 421 kDa, respectively. EbhA shows homology to the major adhesin of Streptococcus defectivus, Emb, a p...

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“…The determination of the role of a given gene in pathogenicity by use of site-directed mutants is hampered by the fact that S. aureus harbors redundant surface adhesins, which might be able to take over and compensate for the function of the missing gene. To circumvent this problem, staphylococcal clfA was recently cloned in Streptococcus gordonii (30) and Lactococcus lactis (27), which lack the redundant adhesive molecules. ClfA was expressed as a surface protein in these bacteria, which thereby gained the ability to cause endocarditis in an animal model (26).…”

mentioning

confidence: 99%

Transcription of Clumping Factor A in Attached and Unattached Staphylococcus aureus In Vitro and during Device-Related Infection

Wolz

Goerke

Landmann³

et al. 2002

Infect Immun

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Staphylococcus aureus is one of the pathogens most frequently isolated in device-related infections. S. aureus is equipped with surface-associated proteins promoting specific binding to matrix molecules. Clumping factor A (ClfA, encoded by clfA) mediates adhesion to fibrinogen. Whereas the contribution of ClfA to pathogenicity is well documented, the influence of different growth and host parameters on gene activity is unclear. To elucidate this question, we investigated clfA transcript levels in an animal model of device-related infection and in planktonic and sessile bacteria grown in vitro. Specific mRNA from the S. aureus strains Newman, Reynolds, and RN6390 was quantified by LightCycler reverse transcription-PCR. In vitro, clfA transcript levels were low in the early logarithmic growth phase, but a clear increase was observed after the late logarithmic phase. Quantities of clfA transcripts were four to six times higher in the planktonic than in the sessile bacterial subpopulations grown to the stationary phase. During infection, in strains Newman and Reynolds levels of clfA transcripts in exudates accumulating in the infected devices were lower than those in the bacteria grown in vitro to stationary phase. clfA mRNA levels in the exudates increased during the initial phase of infection and remained constant after 96 h postinoculation. In contrast to the in vitro results, quantities of clfA transcripts in the unattached bacteria of the exudates never exceeded the level of clfA transcripts in the sessile bacteria attached to glass beads. However, a clear increase in clfA quantities in the sessile bacteria was observed late in infection after 144 h. In conclusion, maximal clfA transcript levels are reached late during growth in vitro and in vivo.

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Study of Staphylococcus aureus Pathogenic Genes by Transfer and Expression in the Less Virulent Organism Streptococcus gordonii

Cited by 42 publications

References 42 publications

Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

Reassessing the Role of Staphylococcus aureus Clumping Factor and Fibronectin-Binding Protein by Expression in Lactococcus lactis

Analysis of Ebh, a 1.1-Megadalton Cell Wall-Associated Fibronectin-Binding Protein of Staphylococcus aureus

Transcription of Clumping Factor A in Attached and Unattached Staphylococcus aureus In Vitro and during Device-Related Infection

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