Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile

Abstract

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract.

Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR‐dCas9

“…Under various conditions of pH, ionic strength, and osmotic pressure, and in the presence of different nutrients such as amino acids, nucleotides, and oligosaccharides, WTAs can shift between an extended and a collapsed conformation (Doyle, McDannel, Streips, Birdsell, & Young, ). Such a dynamic shift helps to regulate the exposure of cell wall peptidoglycan to antimicrobials, especially large molecules such as lytic enzymes, thus rapidly adjusting cell resistance (X. Wu et al, ; X. Wu, Kwon, et al, ).…”

“…S. aureus ATCC 6538 cell wall fragments were isolated as previously reported (Shah, Laaberki, Popham, & Dworkin, ; X. Wu et al, ). Briefly, wild‐type cells grown to OD 600 = 0.6–0.8 were washed with deionized (DI) water and resuspended in 4% sodium dodecyl sulfate (SDS) to a final OD 600 of 1.…”

“…The activity of Lst on isolated cell wall fragments was measured based on a previously reported turbidity assay (X. Wu et al, ; X. Wu, Kwon, et al, ). Briefly, 20 μl of isolated cell wall fragments and 10 μl of Lst (30 μg/ml) were mixed with 170 μl of PBS or TSB, and the turbidity decrease was monitored on a Molecular Devices plate reader at 600 nm for 10 min with 3 s intervals.…”

“…Following 4 hr incubation at 50°C and 15 washes with DI water, the pellet was mixed with 0.1 M NaOH and incubated at room temperature for 16 hr with 120 rpm shaking. The suspension was then centrifuged at 14,000 g for 10 min, and the isolated WTA present in the supernatant was analyzed by polyacrylamide gel electrophoresis (PAGE) as previously reported (X. Wu et al, ).…”

“…However, some bacteria are resistant to lytic enzymes under certain circumstances, such as in the presence of human serum or industrial emulsifiers, although the enzymes retain full catalytic activity under these conditions (Osipovitch, Therrien, & Griswold, ; Proenca et al, ; Proença, Leandro, Garcia, Pimentel, & São‐José, ; J. A. Wu, Kusuma, Mond, & Kokai‐Kun, ; X. Wu, Fraser, Zha, & Dordick, ; X. Wu, Paskaleva, Mehta, Dordick, & Kane, ).…”