The extracytoplasmic function sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis - PubMed (original) (raw)
The extracytoplasmic function sigma factor SigV plays a key role in the original model of lysozyme resistance and virulence of Enterococcus faecalis
André Le Jeune et al. PLoS One. 2010.
Abstract
Background: Enterococcus faecalis is one of the leading agents of nosocomial infections. To cause diseases, pathogens or opportunistic bacteria have to adapt and survive to the defense systems encountered in the host. One of the most important compounds of the host innate defense response against invading microorganisms is lysozyme. It is found in a wide variety of body fluids, as well as in cells of the innate immune system. Lysozyme could act either as a muramidase and/or as a cationic antimicrobial peptide. Like Staphylococcus aureus, E. faecalis is one of the few bacteria that are completely lysozyme resistant.
Results: This study revealed that oatA (O-acetyl transferase) and dlt (D-Alanylation of lipoteicoic acids) genes contribute only partly to the lysozyme resistance of E. faecalis and that a specific transcriptional regulator, the extracytoplasmic function SigV sigma factor plays a key role in this event. Indeed, the sigV single mutant is as sensitive as the oatA/dltA double mutant, and the sigV/oatA/dltA triple mutant displays the highest level of lysozyme sensitivity suggesting synergistic effects of these genes. In S. aureus, mutation of both oatA and dlt genes abolishes completely the lysozyme resistance, whereas this is not the case in E. faecalis. Interestingly SigV does not control neither oatA nor dlt genes. Moreover, the sigV mutants clearly showed a reduced capacity to colonize host tissues, as they are significantly less recovered than the parental JH2-2 strain from organs of mice subjected to intravenous or urinary tract infections.
Conclusions: This work led to the discovery of an original model of lysozyme resistance mechanism which is obviously more complex than those described for other Gram positive pathogens. Moreover, our data provide evidences for a direct link between lysozyme resistance and virulence of E. faecalis.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Structural organization of sigV, oatA, dltA and mprF loci.
Large arrows correspond to the indicated genes. Grey areas show the deleted region by double cross over event and that of sigV locus (harbouring an asterisk) represents the deletion carried out in SAS mutant strain. The solid triangle positioned on EF0031 shows the insertion site of the integrative pUCB300-mprF recombinant plasmid generating the mprF mutant. The putative promoters and terminators are indicated with P and T letters, respectively. Primers used to construct the different mutants are indicated by black arrows, and listed in Table 4.
Figure 2. Susceptibility to lysozyme.
The lysozyme sensitivity of E. faecalis JH2-2 and its derivative mutants was tested on LB agar medium. Overnight cultures were adjusted to OD600 = 1 in physiological water, and diluted up to 10−3. Equal volume of each of the 10−1, 10−2, and 10−3 dilutions are plated on LB solid medium containing large scale range of lysozyme concentrations. (A) Photographs of LB plates after 48 hours incubation under the indicated lysozyme concentrations. (B) The overall results of the lysozyme sensitivity experiments are summarized in this histogram.
Figure 3. Complementation assay under lysozyme treatment.
The parental strain E. faecalis JH2-2 (1), and its derivative mutants Δ_sigV_ (2), SAS pMSP3535 (3), and SAS pMSP3535-sigV (4) were grown on LB solid medium without lysozyme (A) or supplemented with 20 mg/ml of lysozyme (B). Five µl of 10−2 dilution of overnight cultures adjusted to OD600 = 1 in physiological water were plated on solid medium, incubated at 37°C for 48 hours and photographed.
Figure 4. Susceptibility to nisin.
The sensitivity to nisin of E. faecalis JH2-2 (•) and its derivative mutants Δ_sigV_ (▪) and Δ_dltA_ (▴) was tested on GM17 medium (open symbols) or on GM17 supplemented with 2 µg/ml of nisin (solid symbols). The kinetic growth was monitored at OD600 nm. Mean values of three independent experiments are shown and standard deviations are indicated.
Figure 5. Triton X-100-induced autolysis assays.
The autolysis of E. faecalis JH2-2 (○) and its derivative mutants Δ_sigV_ (□), Δ_dltA_ (Δ), Δ_oatA_ (◊), and Δ_sigV/oatA/dltA_ (*) was carried out in Tris-HCl buffer containing 0.1% of Triton X-100. The autolysis was monitored by measuring the decrease in OD600 nm of the cell suspensions every 30 min.
Figure 6. SigV is required for the virulence of E. faecalis in a murine systemic model.
Enterococcal tissue burdens in kidneys (A) and in livers (B) from BALB/c mice infected intravenously with 5×108 cells of E. faecalis JH2-2 wild-type (•) and its isogenic mutant strains Δ_dltA_ (▪), Δ_oat_ (▴), Δ_sigV_ (▾), and Δ_dltA_-_oat_-sigV (♦). Kidney pair and livers homogenates were obtained from groups of 10 mice sacrificed and necropsied at day 7 post-infection. Results, expressed as log10 CFUs per gram of tissue, represent values recorded separately for each of the 10 mice. Horizontal bars represent the geometric means. p value of less than 0.05 was considered to be significant.
Figure 7. SigV is required for the virulence of E. faecalis in a murine UTI model.
Enterococcal burdens of kidneys (A) and bladders (B) of Balb/c mice infected transurethrally with 104 cells of E. faecalis faecalis JH2-2 wild-type (•) and its isogenic mutant strains Δ_dltA_ (▪), Δ_oat_ (▴), Δ_sigV_ (▾), and Δ_dltA_-_oat_-sigV (♦). Kidney pair and bladders homogenates were obtained from groups of 15 mice that were sacrificed and necropsied 48 h after the transurethral challenge. Results, expressed as log10 CFUs per gram of tissue, represent values recorded separately for each mouse. Horizontal bars represent the geometric means. A value of 0 was assigned to uninfected kidneys or bladders. p value of less than 0.05 was considered to be significant.
Figure 8. Comparison between the lysozyme resistance effectors of S. aureus and E. faecalis.
The lowest concentrations of lysozyme resistance are indicated in parenthesis (data for S. aureus are from Herbert et al. [13]) and the most relevant are shaded. The relationship to virulence of the E. faecalis lysozyme resistance effectors is also indicated. Arrows thickness is relatively proportional to the involvement of the considered gene products in lysozyme resistance or virulence. Blocked arrows correspond to no effect on the considered event. Signs + and – correspond to up and down-regulation, respectively.
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