Staphylococcus aureus CidA and LrgA Proteins Exhibit Holin-Like Properties

Abstract

The Staphylococcus aureus cid and lrg operons are known to be involved in biofilm formation by controlling cell lysis and the release of genomic DNA, which ultimately becomes a structural component of the biofilm matrix. Although the molecular mechanisms controlling cell death and lysis are unknown, it has been hypothesized that the cidA and lrgA genes encode holin-and antiholin-like proteins and function to regulate these processes similarly to bacteriophage-induced death and lysis. In this study, we focused on the biochemical and molecular characterization of CidA and LrgA with the goal of testing the holin model. First, membrane fractionation and fluorescent protein fusion studies revealed that CidA and LrgA are membrane-associated proteins. Furthermore, similarly to holins, CidA and LrgA were found to oligomerize into high-molecular-mass complexes whose formation was dependent on disulfide bonds formed between cysteine residues. To determine the function of disulfide bond-dependent oligomerization of CidA, an S. aureus mutant in which the wild-type copy of the cidA gene was replaced with the cysteine mutant allele was generated. As determined by ␤-galactosidase release assays, this mutant exhibited increased cell lysis during stationary phase, suggesting that oligomerization has a negative impact on this process. When analyzed for biofilm development and maturation, this mutant displayed increased biofilm adhesion in a static assay and a greater amount of dead-cell accumulation during biofilm maturation. These studies support the model that CidA and LrgA proteins are bacterial holin-/antiholin-like proteins that function to control cell death and lysis during biofilm development.

Impact of Extracellular Nuclease Production on the Biofilm Phenotype of Staphylococcus aureus under In Vitro and In Vivo Conditions

“…However, most of these studies, including our own, were limited to in vitro experiments that may or may not reflect the more therapeutically relevant in vivo conditions. Extracellular nuclease has also been shown to have a negative impact on biofilm formation (9,13,17,19), and this suggests that exogenous nuclease could be used to limit biofilm formation and thereby reduce the therapeutic recalcitrance of biofilm-associated S. aureus infections. In contrast, nuclease production has also been shown to promote the ability of S. aureus to cause disease by promoting escape from neutrophil extracellular traps (NETs) (4,16).…”

Identification and characterization of HolGH15: the holin of Staphylococcus aureus bacteriophage GH15

“…However, the holin protein of lambda phage 21 forms pinholes that only depolarize the membrane (Pang et al, 2009(Pang et al, , 2010a(Pang et al, , b, 2013. Holins are also important for many activities, such as classification, gene transfer (Lang & Beatty, 2001), biofilm formation (Ranjit et al, 2011) and biotechnology material production (Gao et al, 2013). In contrast to the holins derived from Gram-negative bacterial phages (particularly the lambda, T4 and 21 phages) (Golec et al, 2010;Krupovic & Bamford, 2008;Rydman & Bamford, 2003;Ziedaite et al, 2005), holins encoded by Gram-positive bacterial phages have not been subjected to systematic and in-depth research.…”

Modulation of Staphylococcus aureus Biofilm Matrix by Subinhibitory Concentrations of Clindamycin

“…Autolysis in S. aureus biofilms is also regulated by the activity of the Cid/Lrg holin-antiholin system. CidA oligomerizes in the bacterial cell membrane and increases the activity of murein hydrolases, possibly by exporting them into the extracellular space (22,30,31). In the dispersal phase, bacteria detach from the biofilm and spread to new infection sites.…”