Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosan's activity is postulated. Although we contend that there might not be a single classical target that would explain chitosan's antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

Insights into the Mode of Action of Chitosan as an Antibacterial Compound

Sorting of protein a to the staphylococcal cell wall

Using two monoclonal antibodies, we found subtypes among pneumococcal isolates that are typed as serotype 6A by the quellung reaction. The prevalent subtype bound to both monoclonal antibodies and was labeled here 6Aα, whereas the minor subtype bound to only one monoclonal antibody and was labeled 6Aβ. To determine the biochemical nature of the two serologically defined subtypes, we purified capsular polysaccharides (PSs) from the two subtypes and examined their chemical structures with gas-liquid chromatography and mass spectrometry. The study results for 6Aα PS are consistent with the previously published structure of 6A PS, which is →2) galactose (1→3) glucose (1→3) rhamnose (1→3) ribitol (5→phosphate. In contrast, the 6Aβ PS study results show that its repeating unit is →2) glucose 1 (1→3) glucose 2 (1→3) rhamnose (1→3) ribitol (5→phosphate. We propose to continue referring to 6Aα as serotype 6A but to refer to 6Aβ as serotype 6C. Serotype 6C would thus represent the 91st pneumococcal serotype, with 90 pneumococcal serotypes having previously been recognized. This study also demonstrates that a new serotype may exist within an established and well-characterized serogroup or serotype.

Discovery of a New Capsular Serotype (6C) within Serogroup 6 of Streptococcus pneumoniae

Many surface proteins are thought to be anchored to the cell wall of Gram-positive bacteria via their C-terminus. Cell wall anchoring requires a specific sorting signal, normally located at the predicted C-terminus of surface proteins. Here we show that when placed into the middle of a polypeptide chain, the sorting signal causes the specific cleavage of the precursor as well as the cell wall anchoring of its N-terminal fragment, while the C-terminal fragment remains within the cytoplasm. N-terminal sequencing of the C-terminal cleavage fragment suggests that the cleavage site is located between threonine (T) and glycine (G) of the LPXTG motif, the signature sequence of cell wall sorting signals. All surface proteins harbouring an LPXTG sequence motif may therefore be cleaved and anchored by a universal mechanism. We also propose a novel hypothesis for the cell wall linkage of surface proteins in Gram-positive bacteria.

Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in Gram-positive bacteria

Macrophage scavenger receptors exhibit unusually broad binding specificity for polyanionic ligands and have been implicated in atherosclerosis and various host defense functions. Using a radiolabeled, secreted form of the type I bovine macrophage scavenger receptor in an in vitro binding assay, we have found that this receptor binds to intact Gram-positive bacteria, including Streptococcus pyogenes, Streptococcus agalactiae, Staphylococcus aureus, Enterococcus hirae, and Listeria monocytogenes. Competition binding studies using purified lipoteichoic acid, an anionic polymer expressed on the surface of most Gram-positive bacteria, show that lipoteichoic acids are scavenger receptor ligands and probably mediate binding of the receptor to Gram-positive bacteria. Lipoteichoic acids, for which no host cell receptors have previously been identified, are implicated in the pathogenesis of septic shock due to Gram-positive bacteria. Scavenger receptors may participate in host defense by clearing lipoteichoic acid and/or intact bacteria from tissues and the circulation during Gram-positive sepsis. Since scavenger receptors have been previously shown to bind to and facilitate bloodstream clearance of Gram-negative bacterial endotoxin (lipopolysaccharide), these receptors may provide a general mechanism for macrophage recognition and internalization of pathogens and their cell surface components.

https://www.pnas.org/content/pnas/91/5/1863.full.pdf

The type I macrophage scavenger receptor binds to gram-positive bacteria and recognizes lipoteichoic acid.

A new chemically defined medium for the growth of group A streptococci has been formulated. The advantages of this new medium over previously described defined media are: (i) rates of growth (i.e., doubling times) of 20 strains were comparable to the rates of growth in complex media; (ii) each strain grew to a higher culture density in the new defined medium than in complex media; (iii) transfer from complex media with small inocula was possible without any prior adaptation regimen; and (iv) the production of virulence factors (i.e., M protein and hyaluronic acid) and extracellular enzymes during growth in this new medium was comparable to that in complex media.

https://iai.asm.org/content/iai/27/2/444.full.pdf

Growth characteristics of group A streptococci in a new chemically defined medium.

Protoplasts of a group A streptococcal strain were shown to contain enzymatic activity capable of converting lipoteichoic acid (LTA) to deacylated lipoteichoic acid (dLTA). The enzyme(s) appear to be located mainly in the membrane, although activity was also found in the cytoplasm. Determination of the sites of cleavage within the LTA molecule was approached by comparing the chemical composition of LTA and native dLTA. Native dLTA, as distinguished from chemically deacylated LTA, was isolated from buffer in which live streptococci had been resuspended and incubated. The chemical data suggest that the enzyme(s) was(were) lipolytic in nature; that is, the conversion of LTA to dLTA was the result of cleavage of the ester linkages between the fatty acids and the remainder of the LTA molecule.

/pdf/characterization-and-localization-of-the-enzymatic-uj76t89e1v.pdf

Characterization and localization of the enzymatic deacylation of lipoteichoic acid in group A streptococci.

Changes in the structural components of the Streptococcus pyogenes membrane between exponential and early stationary phases of growth are reported. The overall protein composition ranged from 70 to 73% of the dry weight of the membranes, irrespective of the phase of growth from which they were isolated. Amino acid analyses of membranes isolated from streptococci in either the exponential or stationary phase of growth demonstrated that two amino acids, cysteine and tryptophan, were absent. Further analysis of the membrane proteins by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis demonstrated that there were proteins unique to a particular phase of growth as well as differences in the amount of specific proteins from the various growth phases. In addition, membranes isolated from exponential-phase cultures contained a higher percentage of peripheral protein than did stationary-phase membranes. There also appeared to be an increase in the amount of outer surface proteins during this growth phase. The phosphorus content of the membranes increased during the stationary phase of growth, whereas the sugar composition remained constant. The only sugar found under various conditions of growth in any of the strains was glucose. Total fatty acid content and the mole percent composition of various fatty acids did not change in the different phases of growth. However, the mole percent composition of fatty acids in the membranes of various group A streptococci did differ between strains. Therefore, these results provide evidence that the composition of membranes of S. pyogenes does not remain constant throughout the growth phases of the culture. Images

Chemical Analysis of Changes in Membrane Composition During Growth of Streptococcus pyogenes

The antigenic composition and molecular structure of the plasma membrane of Streptococcus pyogenes (group A; M type 6) were studied by crossed immunoelectrophoresis (XIE) and other related quantitative immunoelectrophoretic techniques. After establishment of a reference pattern of 29 immunoprecipitates, the relative differences in amounts of individual antigens contained in membranes isolated from cells that were harvested during the exponential or stationary phase of growth were examined. Relative increases and decreases in amounts of individual antigens were estimated from the areas subtended by immunoprecipitates after XIE of Triton X-100 extracts. The asymmetric distribution of antigens on the inner and outer surfaces of the membrane was established in absorption experiments with intact, stable protoplasts. Of the 29 immunoprecipitates, 8 appeared to contain antigens exposed on the outer surface of the membrane, whereas 11 appeared to contain antigens either located on the inner surface or unexposed. Six antigens appeared to have limited exposure on the outer surface, and four others remain to be assigned. Certain immunoprecipitates were characterized with respect to enzymatic activity or interaction with the lectin concanavalin A. Reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3), adenosine triphosphatase (EC 3.6.1.3), and polynucleotide phosphorylase (EC 2.3.7.8) were demonstrated by zymogram techniques. The latter two activities were present within the same immunoprecipitate, suggesting the occurrence of a multienzyme complex. In addition, the areas under the immunoprecipitates containing the three enzymatic activities were not affected by absorption of antimembrane immunoglobulin with intact protoplasts and thus appeared to be located on the inner surface of the membrane. The results from absorption experiments also suggested that the exposure of outer protoplast surface antigens was greater on protoplasts from exponential-phase cells than on those from stationary-phase cells, even when found in increased amounts in the latter.

Quantitative immunoelectrophoretic analysis of Streptococcus pyogenes membrane.

The rate of killing of a group A streptococcal species by penicillin was compared with the release of lipoteichoic acid (LTA) and its deacylated derivative, dLTA. Although there was no stimulation of release from stationary-phase cells in the presence of penicillin, there was dramatic release of LTA and dLTA from exponential-phase cells after the addition of penicillin. Although decreases in viability were observed within 15 min after addition of penicillin, culture mass and LTA content did not appear to be affected until after 30 min. Stimulation of release of LTA and dLTA appeared to take place after 15 but before 30 min after addition of penicillin. These observations are interpreted to indicate that the stimulation of release of LTA and dLTA in response to penicillin is secondary to the killing event.

R E Kessler

Papers

Growth characteristics of group A streptococci in a new chemically defined medium.

Characterization and localization of the enzymatic deacylation of lipoteichoic acid in group A streptococci.

Chemical Analysis of Changes in Membrane Composition During Growth of Streptococcus pyogenes

Quantitative immunoelectrophoretic analysis of Streptococcus pyogenes membrane.

Effects of penicillin on group A streptococci: loss of viability appears to precede stimulation of release of lipoteichoic acid.