A reference map of the membrane proteome of <i>Enterococcus faecalis</i>

Maddalo, Gianluca; Chovanec, Peter; Stenberg-Bruzell, Filippa; Nielsen, Hailyn V.; Jensen‐Seaman, Michael I.; Ilag, Leopold L.; Kline, Kimberly; Daley, Daniel O.

doi:10.1002/pmic.201100103

Cited by 18 publications

(30 citation statements)

References 26 publications

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“…This indicates that different cold-shock proteins also able to interact with nucleic acid may be present on the surface. Such types of protein have not been found in the reference map of the membrane proteome of E. faecalis (Maddalo et al, 2011). Nevertheless, in this study, only 102 unique proteins were identified, which may correspond to onlỹ 10 % of the membrane-embedded predicted proteome.…”

Section: Discussionmentioning

confidence: 78%

“…Nevertheless, in this study, only 102 unique proteins were identified, which may correspond to onlỹ 10 % of the membrane-embedded predicted proteome. Interestingly, among them, 29 were characterized as soluble proteins (Maddalo et al, 2011). Identification of a priori cytoplasmic proteins is not unusual and it is well known that some proteins lacking signal sequences or motifs important for their secretion, such as LuxS of Salmonella (Kint et al, 2009), SodA of Listeria (Archambaud et al, 2006) or enolase of E. faecalis (Boël et al, 2004), may have an extracellular location thanks to 'non-classical secretion' (Benachour et al, 2009;Bendtsen et al, 2005;Bøhle et al, 2011).…”

Section: Discussionmentioning

confidence: 99%

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Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis

et al. 2013

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CspR has been characterized recently as a cold-shock RNA-binding protein in Enterococcus faecalis, a natural member of the gastro-intestinal tract capable of switching from a commensal relationship with the host to an important nosocomial pathogen. In addition to its involvement in the cold-shock response, CspR also plays a role in the long-term survival and virulence of E. faecalis. In the present study, we demonstrated that anti-CspR immune rabbit serum protected larvae of Galleria mellonella against a lethal challenge of the WT strain. These results suggested that CspR might have a surface location. This hypothesis was verified by Western blot that showed detection of CspR in the total as well as in the surface protein fraction. In addition, identification of surface polypeptides by proteolytic shaving of intact bacterial cells followed by liquid chromatography-MS-MS revealed that cold-shock proteins (EF1367, EF2939 and CspR) were present on the cell surface. Lastly, anti-CspR immune rabbit serum was used for immunolabelling and detected with colloidal gold-labelled goat anti-rabbit IgG in order to determine the immunolocalization of CspR on E. faecalis WT strain. Electron microscopy images confirmed that the cold-shock protein RNA-binding protein CspR was present in both cytoplasmic and surface parts of the cell. These data strongly suggest that CspR, in addition to being located intracellularly, is also present in the extracellular protein fraction of the cells and has important functions in the infection process of Galleria larvae. INTRODUCTIONEnterococcus faecalis, a natural member of the intestinal tract, is versatile and can switch from a commensal relationship with the host to a leading cause of nosocomial infections (Murray, 1990;Sreeja et al., 2012; Wisplinghoff et al., 2004). This bacterium, well known to be able to cope with hostile environments (Ogier & Serror, 2008) and used as an indicator of faecal contamination, is responsible for serious infections such as endocarditis or surgical wound infection, especially in immunocompromised patients Shepard & Gilmore, 2002). Therefore, the virulence of E. faecalis has been intensively studied over the past 20 years and~12 putative virulence genes have been reported, including several transcriptional and post-transcriptional regulators. The recently identified cold-shock RNA-binding protein CspR is part of this last category of virulence-associated factors (Fox et al., 2009;Hancock & Gilmore, 2002;Lebreton et al., 2009;Michaux et al., 2011 Michaux et al., , 2012Qin et al., 2001; Shankar et al., 2001). In some Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis, cold-shock polypeptides have been shown to be involved in several aspects of bacterial life, i.e. survival under starvation and low-temperature conditions or resistance to anti-microbial agents (Duval et al., 2010;Graumann & Marahiel, 1999, 1996 Graumann et al., 1997). In addition, the cold-shock polypeptides usually present the conserved RNA-binding motifs RNP-1 and RNP-...

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Section: Discussionmentioning

confidence: 78%

Section: Discussionmentioning

confidence: 99%

Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis

et al. 2013

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“…Our 1D SDS/PAGE and membrane shaving protocols resulted in 58 and 25 proteins, respectively that were common to the membrane‐associated proteins identified by Maddalo et al . and Bøhle et al . with 15 proteins common to both protocols.…”

Section: Resultsmentioning

confidence: 91%

“…, Maddalo et al . , and Bøhle et al . for comparison with previous identifications and predicted roles in biofilm formation, stress, and virulence.…”

Section: Methodsmentioning

confidence: 96%

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Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry

et al. 2016

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Enterococcus faecalis is a significant nosocomial pathogen, which is able to survive in diverse environments and resist killing with antimicrobial therapies. The expression of cell membrane proteins play an important role in how bacteria respond to environmental stress. As such, the capacity to identify and study membrane protein expression is critical to our understanding of how specific proteins influence bacterial survival. Here, we describe a combined approach to identify membrane proteins of E. faecalis ATCC V583 using membranes fractionated by either 1D SDS/PAGE or membrane shaving, coupled with LC ‐ ESI mass spectrometry. We identified 222 membrane‐associated proteins, which represent approximately 24% of the predicted membrane‐associated proteome: 170 were isolated using 1D SDS/PAGE and 68 with membrane shaving, with 36 proteins being common to both the techniques. Of the proteins identified by membrane shaving, 97% were membrane‐associated with the majority being integral membrane proteins (89%). Most of the proteins identified with known physiology are involved with transportation across the membrane. The combined 1D SDS/PAGE and membrane shaving approach has produced the greatest number of membrane proteins identified from E. faecalis to date. These protocols will aid future researchers investigating changes in the membrane proteome of E. faecalis by improving our understanding of how E. faecalis adapts and responds to its environment.

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Proteomics of Lactic Acid Bacteria

Lü

2014

Lactic Acid Bacteria

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A reference map of the membrane proteome of Enterococcus faecalis

Cited by 18 publications

References 26 publications

Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis

Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis

Isolation and identification of Enterococcus faecalis membrane proteins using membrane shaving, 1D SDS/PAGE, and mass spectrometry

Proteomics of Lactic Acid Bacteria

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