A novel gene, fudoh, in the SCCmec region suppresses the colony spreading ability and virulence of Staphylococcus aureus - PubMed (original) (raw)

A novel gene, fudoh, in the SCCmec region suppresses the colony spreading ability and virulence of Staphylococcus aureus

Chikara Kaito et al. PLoS One. 2008.

Abstract

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Deletion of the SCC_mec_ region of the MRSA isolates increases colony spreading.

(A) Comparison of colony spreading ability between MSSA and MRSA. Overnight cultures of clinical isolates of MSSA and MRSA were spotted onto soft agar plates and incubated for 10 h at 37°C. Two independent experiments with duplicates were performed and the mean halo diameters are presented as dots. The group means of MSSAs and MRSAs are presented as a bold bar. Statistical analysis was performed with Student's t test. (B) The mecA gene and the partial region of rpoB were amplified by PCR and electrophoresed and stained with ethidium bromide. The rpoB was used as control. (C) Overnight culture of NI-3, NI-4, and NI-5 harboring pND50 or pccrAB (ΔSCC_mec_) was spotted onto soft agar plates and incubated for 10 h. Representative images from three independent experiments are shown. (D) The halo diameter was measured and the means±standard deviations from three independent experiments are presented. Statistical analysis was performed with Student's t test.

Figure 2

Figure 2. A novel gene, fudoh, in the SCCmec region suppresses the colony spreading ability of the Newman strain.

(A) Schematic representation of the mecA, mecR1, mecI, and fudoh genes in the SCC_mec_ region. MRSA chromosomal DNA is represented as a thin line, and the DNA fragment is represented by a thick line, and the deleted DNA region is represented by a dotted line. The boxes above the thin line indicate genes transcribed from left to right, whereas the box below the thin line indicates gene transcribed from right to left. Grey box represents the fudoh gene. (B) The Newman strain was transformed with the integration plasmids described in (A), and colony spreading was examined. The photograph was taken after 10 h incubation. (C) The means±standard deviations of the halo diameters of at least two independent experiments are presented. Statistical analysis was performed with Student’s t test. The _P-_values are versus pInt. NS, not significant (_P_>0.05).

Figure 3

Figure 3. The fudoh gene is mutated in high-spreading MRSA strains.

(A) The fudoh gene of 40 MRSA strains was sequenced. The sequences are organized according to the amount of colony spreading. The amounts of colony spreading are shown in the right bar graph from higher to lower values. The mutated nucleotide residues and the substituted amino acid residue are colored red. (B) The Newman strain was transformed with an integration plasmid harboring either the intact fudoh gene (pInt-fudoh) or a mutated fudoh gene (pInt-K29R-fudoh), and colony spreading was examined. The photograph was taken after 10 h incubation. (C) The means±standard deviations of the halo diameters from three independent experiments are presented.

Figure 4

Figure 4. The fudoh gene decreases S. aureus exotoxin production and virulence in mice.

(A) Hemolytic activities of culture supernatants of the Newman strains transformed with pInt or pInt-fudoh were measured using sheep erythrocytes. The data are means±standard deviations from three independent experiments. (B) Nuclease activities were measured using salmon sperm DNA. (C) CD-1 mice (n = 10) were injected intravenously with a diluted bacterial solution of Newman/pInt or Newman/pInt-fudoh. Statistical analysis was performed with the Kaplan-Meier test. The _P-_value between pInt and pInt-fudoh is less than 0.0001.

Figure 5

Figure 5. Colony spreading of CA-MRSA.

(A) Overnight cultures of CA-MRSA strains were spotted onto soft agar plates and incubated for 10 h at 37°C. The dotted line indicates the averaged value. (B) The DNA fragments containing fudoh and the partial region of rpoB were amplified by PCR using the CA-MRSA genomes, Newman/pInt genome, or Newman/pInt-fudoh genome as the template. The amplified DNA was electrophoresed in 1% agarose gel and stained with ethidium bromide.

Similar articles

Cited by

References

    1. Hiramatsu K. Vancomycin-resistant Staphylococcus aureus: a new model of antibiotic resistance. Lancet Infect Dis. 2001;1:147–155. - PubMed
    1. Chambers HF. Community-associated MRSA–resistance and virulence converge. N Engl J Med. 2005;352:1485–1487. - PubMed
    1. Rozgonyi F, Kocsis E, Kristof K, Nagy K. Is MRSA more virulent than MSSA? Clin Microbiol Infect. 2007;13:843–845. - PubMed
    1. Mizobuchi S, Minami J, Jin F, Matsushita O, Okabe A. Comparison of the virulence of methicillin-resistant and methicillin-sensitive Staphylococcus aureus. Microbiol Immunol. 1994;38:599–605. - PubMed
    1. Voyich JM, Braughton KR, Sturdevant DE, Whitney AR, Said-Salim B, et al. Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J Immunol. 2005;175:3907–3919. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources