Absence of autolytic activity (peptidoglycan nicking) in penicillin-induced nonlytic death in a group A streptococcus

Abstract

The extent of sublytic autolysin activity (peptidoglycan [PG] nicking) after exposure of exponentially growing cultures of a group A streptococcus (GAS) to benzylpenicillin (PenG) was studied by determining changes in the glycan chain length of PG polymers. The average PG chain length in isolated cell walis was estimated by calculating the ratio of the total hexosamine content (Morgan-Elson-reactive material) A unified model for the action of inhibitors of cell wall synthesis (21, 26) predicts that in all susceptible organisms, cell wall antibiotics initially act by inhibiting the assembly of insoluble peptidoglycan (PG), which in turn leads to bacteriostasis. The subsequent (secondary) events are species dependent and, on the basis of recent reports, can also be related to growth rate (4) and medium composition (14). Apparently, some bacteria die and then lyse, whereas others lyse immediately, and still others undergo nonlytic death. An additional response, tolerance, is considered in the model as a continuation of stasis (21) and has been suggested to be associated with specific regulatory mechanisms (19,21). A central feature of lytic phenotypes is the secondary expression of endogenous PG hydrolases (autolysins) after exposure to antibiotics (21,26). It follows that a prerequisite for nonlytic phenotypes is an absent, weakly expressed, or tightly regulated autolytic system. PG hydrolase activities are considered to be essential for normal surface growth and cell division processes (5) and are therefore presumed to be present in all bacterial cells. Considering the following phenomena: (i) the demonstrated unity in the primary mechanism of action of, for example, P-lactams in the binding to penicillin-binding proteins and inhibition of their function(s) (2, 9), (ii) the requirement of autolytic activities for growth (5), ahd (iii) the documented relationship between inhibition of PG assembly and deregulation of autolysins in lytic phenotypes (21, 26), hydrolysis of a small number of bonds insufficient to cause cellular lysis (nicking) has been suggested as a possible secondary and potentially fatal outcome of inhibition of PG synthesis in bacteria which undergo nonlytic death (8).In this report, we present results of experiments designed to assess the role of PG nicking in penicillin-induced nonlytic death in a group A streptococcus (GAS). Preliminary characterization of the autolytic system of GAS is also presented.( Quantitative comparison of growth inhibition. Conventional methods for determining MICs were considered to be unsatisfactory for use in these studies, since they rely on small, physiologically poorly defined inocula and long incubation intervals. Therefore, 50% growth inhibitory concentrations (GIC50s) were determined for each strain as described previously (17). For these studies we used GIC50s obtained after exposure of exponentially growing cultures at the time that exponentially growing control cultures had increased fourfold in turbidity. Potassium PenG (1,595 U/mg) used in these experim...

Two bactericidal targets for penicillin in pneumococci: autolysis-dependent and autolysis-independent killing mechanisms

“…The concurrence of cell death and lysis in these bacteria has led to the widely held belief that the cause of cell death is the suicidal (unregulated) activity of autolytic enzymes. On the other hand, it is also known that in certain species of bacteria penicillin can have powerful bactericidal activity without accompanying autolysis (10,13,18), suggesting that mechanisms other than cell lysis may exist in bacteria for the killing effect of penicillin.In order to clarify the relationship between penicillininduced lysis and killing in pneumococci, we performed a series of experiments using autolysis-inhibiting agents and pneumococcal autolysin mutants carrying a variety of defects in the structural determinant (IytA) of the major autolysin (an N-acetylmuramoyl-L-alanine amidase; referred to as amidase). We also characterized in detail a recently described (19) new type of mutation (cid) that drastically reduces penicillin-induced killing in both wild-type (Lyt+) cells and Lyt-mutants of pneumococci.…”

Loss of Antibiotic Tolerance in Sod-Deficient Mutants Is Dependent on the Energy Source and Arginine Catabolism in Enterococci

“…On the basis of previous studies, it was suggested that the tolerance might be due to alterations in bacterial autolysis, because killing of nontolerant bacteria was often accompanied by massive cell lysis (6). However, penicillin has been shown to kill pathogens such as Streptococcus pyogenes and Enterococcus hirae efficiently without inducing significant lysis of the cells (7)(8)(9). Furthermore, suppression of autolysis in Streptococcus pneumoniae and Staphylococcus aureus had no significant effect on killing by penicillin (6,(10)(11)(12)(13).…”

Deregulation of the Arginine Deiminase ( arc ) Operon in Penicillin-Tolerant Mutants of Streptococcus gordonii

“…Moreover, unlike pneumococci, S. gordonii is not lysed during beta-lactam therapy. This allows an analysis that is more restricted to autolysis-independent mechanisms of penicillin-induced killing and penicillin tolerance (13,25).…”

“…However, several studies indicated that autolysis is not the sole mechanism of penicillin-induced killing. Certain bacteria, such as Streptococcus pyogenes and other gram-positive cocci, are killed extensively by penicillin in spite of the fact that they are not lysed by the antibiotic (6,14,25). Moreover, specific blockage of penicillin-induced lysis in Streptococcus pneumoniae and Staphylococcus aureus decreased drug-induced killing only marginally, thus suggesting that other, autolysis-independent pathways were involved in penicillin-induced lethality (11,13,26,31,36,37).…”