Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis - PubMed (original) (raw)
Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis
Sreedhar R Nallapareddy et al. Infect Immun. 2008 Sep.
Abstract
Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P <or= 0.0002), making Acm the first factor shown to be important for E. faecium pathogenesis. In contrast, no mortality differences were observed in a mouse peritonitis model. While 5 of 17 endocarditis isolates were Acm nonproducers and failed to adhere to collagen in vitro, all had an intact, highly conserved acm locus. Highly reduced acm mRNA levels (>or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.
Figures
FIG. 1.
Attenuation of an acm deletion mutant in a rat endocarditis model. (A) Percentages of wild-type TX0082 and the acm deletion mutant TX6051 recovered from the initial inocula (left) and from vegetations 72 h after infection of 14 rats (right) are shown. In 10 rats, zero colonies in the vegetation were mutants and were assigned individualized minimum detection values (see the text for details). Horizontal lines indicate means (P < 0.0001 [by paired t test] for percentage of total bacteria in the vegetation versus that in the inoculum). (B) Log10 CFU/g of vegetations from rats infected with a mixture of wild-type E. faecium TX0082 and its isogenic acm deletion mutant TX6051. The horizontal line indicates the geometric mean. Empty circles and triangles represent inoculum percentages, and solid circles and triangles represent vegetation or valve percentages of wild-type TX0082 and TX6051, respectively. The single rat in which the mutant outnumbered the wild type is shaded in gray.
FIG. 2.
The acm mutant TX6051 is defective in colonization of damaged aortic valves. The percentages of wild-type TX0082 and the acm deletion mutant TX6051 recovered from heart valves 1 h and 3 h after infection of 13 rats and from the initial inocula are shown. Horizontal lines indicate means (P < 0.0001 for 1 h and P = 0.0002 for 3 h versus 0 h). Empty circles and triangles represent inoculum percentages, and solid circles and triangles represent vegetation or valve percentages of wild-type TX0082 and TX6051, respectively.
FIG. 3.
Analysis of acm transcripts from E. faecium strains. (A) Semiquantitative RT-PCR analysis of acm, using RNAs extracted from three collagen-adhering and two nonadhering E. faecium endocarditis isolates. Results are presented as amplification products electrophoresed in ethidium bromide-stained agarose gels. The first and fifth lanes for each strain represent 250 ng of total mRNA, and lanes 2 to 4 and 6 to 8 show 50, 10, and 2 ng (fivefold serial dilutions) of RNA before RT-PCR. The genomic DNAs (lane 10) used as a control for PCR were extracted from the respective isolates. RT-PCRs performed with two independent sample preparations showed similar results. (B) Relative quantitation of acm expression by real-time RT-PCR. acm transcripts (means ± standard deviations) were quantitated from one collagen-adhering and five nonadhering E. faecium endocarditis strains relative to transcripts of TX0074, a strain that showed surface Acm on >99% of cells (20). 23S rRNA transcripts were used as an endogenous control. The percentages of cells adhering to collagen, the percentages of cells expressing Acm on their surfaces in vitro, and anti-Acm titers of the respective infected patient sera from the companion study (20) are given at the bottom of the figure.
FIG. 4.
Western blots of mutanolysin surface protein extracts of E. faecium isolates probed with anti-Acm A-domain-specific Igs. Ten micrograms of mutanolysin extracts of E. faecium isolates and 10 ng of recombinant Acm A domain were used for each lane. TX0074 and TX0082 are adherent strains, while TX0016, TX0068, TX0080, TX0110, TX0111, and TX6051 are nonadherent strains.
FIG. 5.
FACS analysis of Acm surface expression in E. faecium cells. (A) Analysis of Acm surface expression in E. faecium TX0016 cells after sodium _m_-periodate treatment. Cells were incubated with control PRS Igs or anti-Acm A-domain-specific Igs, followed by incubation with F(ab′)2 fragments of goat anti-rabbit IgG (H+L) conjugated to _R_-phycoerythrin (antibody II). (B) Flow cytometry analysis of Acm surface expression in E. faecium cells derived from vegetations of rat experimental IE. Processed vegetations were incubated with control PRS Igs, anti-Acm A-domain-specific Igs, or Igs purified from antiserum raised against formalin-killed TX0016 whole cells (anti-TX0016), followed by incubation with antibody II. Specific binding by anti-Acm or anti-TX0016 antibodies is indicated as log fluorescence intensity on the x axis. Each histogram represents 50,000 bacteria (A) or 10,000 to 38,000 events (bacterium-sized particles) (B).
FIG. 6.
Inhibition of adherence of E. faecium strain TX0082 to collagen type I by Acm A-specific Igs eluted from E. faecium endocarditis patient serum. 35S-labeled bacteria were incubated with 1 or 5 micrograms/ml of eluted Acm A-specific antibodies for 1 h at 37°C. Adherence was tested in wells coated with 1 μg of collagen. Bars represent the percentages of cells bound (means ± standard deviations) for 6 to 12 wells. Results are representative of three independent experiments. NHS Ig: total Igs from NHS from healthy volunteers; HFmEPS anti-Acm Ig, _h_uman _E. faecium e_ndocarditis _p_atient _s_erum-derived specific anti-Acm Igs eluted against the recombinant Acm A domain; PRS Ig, total Igs from PRS; rabbit anti-Acm37 Ig, specific anti-Acm Igs from rabbit antisera raised against the Acm A domain and eluted against the high-affinity binding segment, rAcm37 (17).
References
- Angrist, A. A., and M. Oka. 1963. Pathogenesis of bacterial endocarditis. JAMA 183249-252. -PubMed
- Bayer, A. S., A. F. Bolger, K. A. Taubert, W. Wilson, J. Steckelberg, A. W. Karchmer, M. Levison, H. F. Chambers, A. S. Dajani, M. H. Gewitz, J. W. Newburger, M. A. Gerber, S. T. Shulman, T. J. Pallasch, T. W. Gage, and P. Ferrieri. 1998. Diagnosis and management of infective endocarditis and its complications. Circulation 982936-2948. -PubMed
- Elasri, M. O., J. R. Thomas, R. A. Skinner, J. S. Blevins, K. E. Beenken, C. L. Nelson, and M. S. Smeltzer. 2002. Staphylococcus aureus collagen adhesin contributes to the pathogenesis of osteomyelitis. Bone 30275-280. -PubMed
- Giannitsioti, E., I. Skiadas, A. Antoniadou, S. Tsiodras, K. Kanavos, H. Triantafyllidi, and H. Giamarellou. 2007. Nosocomial vs. community-acquired infective endocarditis in Greece: changing epidemiological profile and mortality risk. Clin. Microbiol. Infect. 13763-769. -PubMed
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