However, CL depletion had no effect on susceptibility to the antimicrobial peptides ASABF-α and nisin. It is possible that the net negative charge is compensated for by other membrane components such as mTOR inhibitor PG. In fact, the PG level was increased in the mutants that did not

accumulate CL. The importance of positively MM-102 charged lysyl-phosphatidylglycerol (LPG) (or MprF protein) in resistance to cationic antimicrobial peptides has been reported [43, 44], and the LPG level was not different between wild-type S. aureus and cls mutants. In addition to the probable effect of cell surface charge, we have previously reported that cell wall thickness is an important factor affecting resistance to the antimicrobial peptide ASABF-α [33]. Furthermore, in the present study, ASABF-α-resistant strains had cell walls that interfered with CL extraction (Additional file 1, Figure S1). Cell wall thickness may also be related to resistance

against other antimicrobial peptides in S. aureus [45, 46]. Our data indicate that lysostaphin treatment is critical for the efficient extraction of CL from S. aureus. Previous reports have suggested that CL is not readily extractable from B. subtilis and other Gram-positive bacteria without lysozyme treatment [47]. This may be attributable to its large molecular mass (~1500 Da) relative to that of other phospholipids, owing to its four acyl residues. However, ~25-kDa globular hydrophilic molecules can pass freely through the ~2-nm holes in the peptidoglycan polymer that forms the cell wall of Gram-positive bacteria [48]. Instead, the efficiency Epacadostat concentration Meloxicam of CL extraction is likely reduced by its physical interactions with cell wall components; for example, when CL is bound to cell wall components, it will not efficiently enter the organic phase during extraction. The membrane of the L-form variants of S. aureus is thought to express certain features that support cell growth and survival in the absence of a rigid cell wall. One study reported that a particular L-form strain had an increased CL level [36].

Our data demonstrate that the cls2 gene is important for normal L-form generation. However, the cls1/cls2 double mutant still produced L-form cells, suggesting the existence of a CL-independent mechanism. Thus, multiple mechanisms may function in cooperation to generate L-form variants. The production of a number of factors such as carotenoids, catalase, coagulase, lipase, fibrinolysin, hemolysin, and enterotoxin changes upon L-form generation and reversion [49–52]. However, none of these represents a common L-form variant phenotype, suggesting that L-form generation is associated with a drastic phenotypic conversion. The increase in CL content may be important, but not essential, for membrane stabilization. In this study, both cls1 and cls2 were shown to encode functional CL synthases.