Gelatinase is important for translocation of Enterococcus faecalis across polarized human enterocyte-like T84 cells.
Previously, in our laboratory, we established a two-chamber system to study translocation of Enterococcus faecalis across monolayers of polarized human colon carcinoma-derived T84 cells. By using the same system in the present study, we now show that disruption of gelE of strain OG1RF, which also has a polar effect on the cotranscribed sprE, as well as disruption of its regulatory system (fsrA, fsrB, and fsrC) resulted in a loss of detectable translocation by E. faecalis OG1RF; these mutants lost gelatinase (GelE) and serine protease (SprE) production by standard assay. A gelE deletion mutant of OG1RF (GelE- SprE+) also showed that significantly reduced translocation and complementation with the gelE gene (pTEX5438) in trans restored gelatinase and translocation, demonstrating that gelatinase is important for E. faecalis translocation. Complementation of fsrA, fsrB, and fsrC mutants with all three fsr genes also resulted in production of gelatinase and translocation. Furthermore, introduction of fsr genes into two non-gelatinase-producing E. faecalis isolates, the well-characterized laboratory strain JH2-2 and a human-derived fecal isolate, TX1322 (both of which have gelE but not fsrA or fsrB, are gelatinase negative, and do not translocate), resulted in gelatinase production by these strains and restored translocation across T84 monolayers, while transformation with pTEX5438 (gelE) showed little or no translocation and no detectable gelatinase, confirming the importance of both fsr and gelatinase for E. faecalis translocation. The importance of gelatinase production was also corroborated among 20 E. faecalis human isolates (7 fecal, 7 endocarditis, and 6 urine isolates), which showed translocation by all gelatinase-positive isolates but little to no translocation for gelatinase nonproducers. These results indicate that gelatinase is important for the successful in vitro translocation of E. faecalis across human enterocyte-like T84 cells.
Zeng
Jing
J
Department of Internal Medicine, Division of Infectious Diseases, 1728 JFB, University of Texas Medical School, 6431 Fannin St., Houston, TX 77030, USA.
Teng
Fang
F
Murray
Barbara E
BE
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018988:Bacterial Translocation; D045744:Cell Line, Tumor; D016764:Cell Polarity; D004697:Endocarditis, Bacterial / Q000382:microbiology; D013293:Enterococcus faecalis / Q000201:enzymology / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D020895:Enterocytes / Q000382:microbiology*; D005243:Feces / Q000382:microbiology; D018093:Gelatinases / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D006801:Humans; D014556:Urine / Q000382:microbiology
D001426:Bacterial Proteins; D018093:Gelatinases
J Bacteriol. 2005 Jan;187(1):266-75
15601711
Crit Care Med. 2004 Feb;32(2):467-71
14758165
Appl Environ Microbiol. 2002 Jun;68(6):3152-5
12039782
J Clin Microbiol. 2004 Mar;42(3):1149-54
15004067
J Bacteriol. 1964 Sep;88:653-9
14208503
J Infect Dis. 1998 Nov;178(5):1416-20
9780263
Infect Immun. 2000 May;68(5):2579-86
10768947
J Bacteriol. 2001 Jun;183(11):3372-82
11344145
J Biol Chem. 1989 Feb 25;264(6):3325-34
2536744
J Infect Dis. 1990 Jul;162(1):82-90
2113080
Infect Immun. 2000 Feb;68(2):815-23
10639451
Infect Immun. 2003 Oct;71(10):5682-9
14500489
J Clin Microbiol. 2004 May;42(5):2317-20
15131223
J Bacteriol. 1955 Jun;69(6):728-32
14392135
Infect Immun. 2004 Jun;72(6):3628-33
15155673
J Bacteriol. 2003 Jun;185(12):3613-23
12775699
Gene. 1991 Jun 15;102(1):99-104
1864514
J Bacteriol. 1974 Feb;117(2):360-72
4204433
Biochem Biophys Res Commun. 1994 Apr 29;200(2):981-5
8179636
Infect Immun. 1991 Jan;59(1):415-20
1846126
Prep Biochem. 1971;1(4):269-82
5005724
Infect Immun. 2000 Oct;68(10):6044-7
10992519
Dig Dis Sci. 1993 Aug;38(8):1530-6
8344112
Proc Assoc Am Physicians. 1999 Jul-Aug;111(4):328-34
10417741
Clin Microbiol Rev. 1990 Jan;3(1):46-65
2404568
Infect Immun. 2002 Oct;70(10):5647-50
12228293
Mol Microbiol. 2001 Jul;41(1):145-54
11454207
J Appl Microbiol. 2004;96(2):230-43
14723684
J Bacteriol. 1964 Sep;88:586-90
14208492
Bacteriol Rev. 1937 Dec;1(1):3-97
16350049
Antimicrob Agents Chemother. 1998 Nov;42(11):2883-8
9797220