SalB inactivation modulates culture supernatant exoproteins and affects autolysis and viability in Enterococcus faecalis OG1RF

Abstract

The culture supernatant fraction of an Enterococcus faecalis gelE mutant of strain OG1RF contained elevated levels of the secreted antigen SalB. Using differential fluorescence gel electrophoresis (DIGE) the salB mutant was shown to possess a unique complement of exoproteins. Differentially abundant exoproteins were identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Stress-related proteins including DnaK, Dps family protein, SOD, and NADH peroxidase were present in greater quantity in the OG1RF salB mutant culture supernatant. Moreover, several proteins involved in cell wall synthesis and cell division, including d-Ala-d-Lac ligase and EzrA, were present in reduced quantity in OG1RF salB relative to the parent strain. The salB mutant displayed reduced viability and anomalous cell division, and these phenotypes were exacerbated in a gelE salB double mutant. An epistatic relationship between gelE and salB was not identified with respect to increased autolysis and cell morphological changes observed in the salB mutant. SalB was purified as a six-histidine-tagged protein to investigate peptidoglycan hydrolytic activity; however, activity was not evident. High-pressure liquid chromatography (HPLC) analysis of reduced muropeptides from peptidoglycan digested with mutanolysin revealed that the salB mutant and OG1RF were indistinguishable.

Fig 1

Culture supernatant exoproteins from E. faecalis strains. Exoproteins of OG1RF and its isogenic mutants with mutations in salB (Liv729 [OG1RF salB]), gelE (OG1RF gelE), salB gelE (Liv1016 [OG1RF salB gelE]), and salB comp (Liv883 [OG1RF salB pAT18::salB]), harvested at early stationary (8 h) growth phase. Molecular mass markers (kDa) are shown on the left. The arrowhead indicates SalB.

Fig 2

(A) DIGE analysis of culture supernatant exoproteins produced after 8 h of growth by strain Liv729 (OG1RF salB), labeled with Cy3 (yellow-green), and those produced by its parent strain OG1RF, labeled with Cy5 (red). (B) Exoproteins produced by strain Liv729 (OG1RF salB) and separated by 2D SDS-PAGE, with arrowed letters indicating differentially regulated proteins.

Fig 3

Autolysis of E. faecalis strains after addition of penicillin G. Strains are OG1RF (open circles), TX5264 (OG1RF gelE) (filled squares), Liv729 (OG1RF salB) (filled triangles), and Liv1016 (OG1RF gelE salB) (open squares). Error bars represent standard errors of the means from triplicate experiments. Statistically significant differences (P < 0.05) in autolysis were determined at 2 h, 3 h, and 4 h for Liv729 relative to OG1RF and for Liv1016 relative to TX5264, using Student's t test.

Fig 4

Analysis of viability in cell populations using fluorescence-activated cell sorting (FACS) analysis. Cells were labeled using the Live/Dead viability assay kit (Invitrogen), and 100,000 events were recorded. OG1RF (A), TX5264 (OG1RF gelE) (B), Liv729 (OG1RF salB) (C), Liv1016 (OG1RF salB gelE) (D), and Liv883 (OG1RF salB pAT18::salB) (E) were harvested at exponential (5 h) growth phase. Horizontal lines mark populations of dead cells.

Fig 5

Cell morphology and viability determination of E. faecalis strains. Cells of strains TX4002 OG1RF (A), TX5264 (OG1RF gelE) (B), Liv729 (OG1RF salB) (C), and Liv1016 (OG1RF gelE salB) (D) were labeled with BacLight live/dead stain and visualized using laser scanning microscopy. Bars indicate the scales, which vary among the images due to varied morphology (1, 5, 2, and 10 μm, respectively, for panels A, B, C, and D).

Fig 6

Transmission electron microscopy of thin sections of mid-exponentially growing cells of strains TX5244 (OG1RF) (A), Liv729 (OG1RF salB) (B), and Liv1016 (OG1RF gelE salB) (C). Scale bar, 500 nm.

Fig 7

Separation of soluble muropeptides by HPLC after incubation with enzymes. OG1RF peptidoglycan was incubated overnight with 5 μg of mutanolysin (Sigma) (A) or 50 μg of SalB (B) or without enzyme (control) (C).