@proceedings {2204,
	title = {The role of Ieucocyte and serum factors and of cationic polyelectrolytes in the lysis andbiodeg radation of Staphylococcus aureus: relation to the pathogenesis of staphylococcal infections},
	journal = {STAPHYLOCOCCI AND STAPHYLOCOCCAL INFECTIONS},
	year = {1983},
	pages = {325-355},
	address = {London},
	abstract = {INTRODUCTION. Although much is known today about the mechanisms by which
virulent Staphylococcus aureus induce tissue lesions and cause clinical
manifestations in mammals, our knowledge of the role played by {\textquotedblleft}professional{\textquotedblright} phagocytes in host and parasite interrelationships in staphylococcal infections, is not fully understood. The extensive literature
on staphylococci and their role in human disease has been the subject of several excellent comprehensive reviews (Whipple, 1965; Cohen,
1972; Jeljaszewicz, 1976).
It is accepted that the interception of staphylococci by granulocytes
(PMNs) takes place soon after the penetration of the cocci into the tissues.
This involves the release of chemotactic factors, by the bacteria themselves
(Pusell et al., 1975) or the activation, by staphylococcal factors, of
chemotactic agents from complement (Ginsburg and Quie, 1980). Subsequent phagocytosis is markedly enhanced by opsonins (Koenig, 1972; Ekstadt, 1974), and in most cases the engulfed bacteria may be killed
intracellularly by a variety of bactericidal agents generated by activated
PMNs (Klebanoff, 1972). It is also suggested that certain lysosomal
enzymes, which are released into the phagolysosome, may digest the
staphylococcal cells (Cohn, 1963a; De Voe et al., 1973; Ginsburg and
Sela 1976; Ginsburg, 1979) (see Sections II.B and VI). Several reports
have, however, shown that intracellular staphylococci may sometimes
survive within PMNs, where they multiply and eventually kill the cell
(Koenig, 1972; Cohen, 1972; Pearce et al., 1976). Surprisingly, very
little is known about the fate and mechanism of biodegradation of staphylococcal cell constituents, once they have been sequestered within
the phagolysosomes of leucocytes. The importance of this field of research stems from the findings that non-biodegradable cell wall
components of a variety of microbial species may persist within
macrophges for prolonged periods, to trigger chronic infiammatory sequelae (Dannenberg, 1968; Kanai and Kondo, 1974; Ginsburg et al.,
1975a; 1975b; 1976a; Ginsburg and Sela, 1976; Adams, 1976; Page et
al., 1978; Ginsburg, 1979).
The inability of {\textquotedblleft}professional{\textquotedblright} phagocytes to degrade intracellular
bacteria may be due (1) to the presence, on the surface of certain
microorganisms, of shielding capsular material (Dossett et al., 1969;
Smith, 1977; Wilkinson, et al., 1979; Densen and Mandell, 1980),
(2) to the lack of fusion between lysosomes and phagosomes (Goren
et al., 1976; Densen and Mandell, 1980), (3) to the production of
leucocidins (Gladstone and Van Heyningen, 1957; Woodin, 1960;
Ginsburg, 1970; Van Heyningen, 1970; Bernheimer. 1970; Ginsburg,
1972), (4) to the lack of adequate lysosomal enzymes capable of
cleaving bacterial peptidoglycans (Dannenberg, 1968; Ginsburg, 1972;
Ginsburg and Sela, 1976; Page et al., 1978; Ginsburg, 1979), or (5) to
the presence, in serum and in inflammatory exudates, of agents which
interfere with the interaction of bactericidal and bacteriolytic age
with engulfed bacteria. These fields were comprehensively reviewed EW{\textquoteright}
Jeljaszewicz, 1976; Smith, 1977; Ginsburg, 1979; and by Densen and
Mandell, 1980.
During the last 8 years our laboratory has been studying the host-
and-parasite interrelationships in streptococcal and staphylococcal
infections using biochemical, electron microscopical and tissue culture
techniques. In particular we studied the mechanisms by which leu-
cocytes and their isolated lysosomal agents bring about the degrada-
tion of staphylococcal cell wall components, and the role which may
be played by such degradation products in the initiation of chronic
inflammation. The present report is an updated overview of these
studies.},
	author = {Isaac Ginsburg and Michael N. Sela and N. Neeman and Meir Lahav}
}
@proceedings {2201,
	title = {CATIONIC POLYELECTROLYTES ACTIVATE AUTOLYTIC WALL ENZYMES IN STAPHYLOCOCCUSAUREUS: Modulation by anionic polyelectrolytes in relation to the survivalof bacterial constituents in tissues},
	journal = {The Target of Penicillin},
	year = {1983},
	pages = {341-346},
	abstract = {Introduction. 
Although a wealth of knowledge exists today on the biochemical pathways of
biosynthesis, turnover and autolysis of bacterial cell wall components in
vitro (1, 2), surprisingly very little is actually known about the
mechanisms of biodegradation of microbial constituents in_vivo. One should
differentiate between bactericidal and bacteriolytic processes induced by
leukocytes since killed, but non-degraded, microbial cells may persist
within macrophages to trigger chronic inflammation (3, 4). The present
communication further supports our contention (5, 6) that the degradation
of microbial cell wall components by leukocytes may be due to activation,
by leukocytic cationic proteins, of autolytic wall enzymes rather than to
the direct cleavage of the cells by lysosomal hydrolases. The modulation
of bacteriolysis by anionic polyelectrolytes will be described and
discussed in relation to the pathogenesis of chronic inflammation and
afequelae.

},
	author = {Isaac Ginsburg and Meir Lahav}
}
@proceedings {2200,
	title = {INDUCED AUTOLYTIC WALL PROCESSES IN HEAT-INACTIVATEDSTAPHYLOCOCCUS AUREUS},
	journal = {The Target of Penicillin},
	year = {1983},
	pages = {335-340},
	abstract = {Introduction. 
In previous studies it could be shown that autolytic wall enzymes of bacteria can be activated by some cationic proteins (1). Recently we determination of firming our data lytic enzyme but obtained results from chemical and end group determination of the cleavage products from peptidoglycan confirming our data that even lysozyme acted not only as a muralytic enzyme but also as a cationic protein (2). In order to
elucidate the mechanisms of the activation of autolytic wall processes by cationic proteins and of the direct respectively indirect muralytic actions of lysozyme we performed investigations on heated cells.
},
	author = {Meir Lahav and Isaac Ginsburg}
}
@article {1685,
	title = {Lysis and biodegradation of microorganisms in infectious sites may involve cooperation between leukocyte, serum factors and bacterial wall autolysins: A working hypothesis},
	journal = {European Journal of Clinical Microbiology},
	volume = {2},
	number = {3},
	year = {1983},
	pages = {186-191},
	abstract = {Although a voluminous literature exists today
on the mechanisms by which "professional"
leukocytes (granulocytes and maerophages)
intercept with, engulf and eventually kill
phagocytosed microorganisms ~ (1, 2), surprisingly
very little is known about the mechanisms
of degradation and elimination of bacteria
from tissues. It is well established that phagocytic
cells are endowed with numerous hydrolyric
enzymes, including the key cell wall
splitting enzyme-lysozyme, which can theoretically
cleave, surface, eeU wall and cytoplasmic
constituents of bacteria. Also, fresh mammalian
sera are known to contain a complex mixture
of heat-labile complement components (3)
heat-stable lysozyme and platelet-derived cationic
proteins (~-lysins) (4) which have been
shown to kill and partially lyse certain microbial
constituents. Surprisingly, however, the
majority of virulent microorganisms are highly
refractory to both leukocyte and serum lyric
agents. Throughout this communication we
shall use the general term bacteriolysis to denote
the degradation of the cell walls, the outer
membranes and the cytoplasmic constituents
of bacteria. The term cell wall lysis will be used
to describe the specific biochemical degradation
of the bacterial peptidoglycan. This may
also be accompanied by the rupture of the
protoplast membrane and the release of cytoplasmic
constituents.
The inability of leukocyte and serum factors
to induce bacteriolysis is linked to the presence,
upon most bacterial surfaces, of{\textquoteright}lipopolysaccharides,
polysaccharides-teichoic complexes
and certain lipids and waxes, which hinder the accessibility of the major cell wall
splitting enzyme-lysozyme to the peptidoglycan
(1). Once however this obstacle is overcome,
the peptidoglycan ist degraded, and the
protoplasts burst due to their high osmotic
pressure, releasing degradation products of
both cell wall and cytoplasmic constituents
into the surrounding medium. The very extensive
literature on these subjects has been recently
summarized and reviewed (5-7).
It has als0 been suggested that the process of
killing and biochemical degradation of microbial
constituents, either following phagocytosis
or following treatment with fresh complement
and lysozyme-sufficient serum are probably
mediated by different mechanisms (6-8). While
extensive loss of wall material and cytoplasmic
entities is usually accompanied by a bactericidal
reaction, the killing of bacteria either by the
oxygen-dependent (9) or by the non-oxygen
dependent bactericidal systems of leukocytes
(5, 10) and serum (3), is not necessarily accompanied
by a substantial bacteriolysis. The distinction
between a bactericidal and a bacteriolyric
process is important, in view of the observations
that poorly degraded non-viable
microbial constituents may persist for long
periods both extracellularly and within phagocytic
cells, to trigger and perpetuate chronic
inflammatory sequellae (6, 7, 11-13). Furthermore,
while degradation products of grampositive
and acid-fast bacteria have been shown
to be endowed with distinct pharmacological
properties (14), to modulate the immune responses
(15), to activate the complement
cascade (16) and to be cytopathic for mammalian
cells (14), the in vivo release of lipopolysaccharides
of the outer membrane of gramnegative
bacteria may result in severe coagulation
defects (Shwartzman phenomenon) and
in endotoxic shock (17). Poorly degraded cell
wall components of bacteria have also been
shown to be translocated within macrophages
from one tissue site to another, thus contributing perhaps to the dissemination of granulomatosis
(18-20).
Although the nature of the biochemical pathways
involved in bacterial biodegradation in
tissues has not been fully elucidated, it has
been recently suggested that a cooperation
among leukocyte factors (21, 22), serum components
(23), the bacterial own autolytic wall
enzymes (21, 22) and certain antibiotics (24),
may act in accord to induce a massive breakdown
of cell wall and cytoplasmic constituents
of bacteria.
It is well established that the autolytic systems
present in every bacterial cell, control cell division,
the deposition of new cell wall material
and the regeneration after treatment with certain
antibiotics (25, 36). Autolytic enzymes
have been isolated from many bacterial species
and were found to possess muramidase, Nacetyl
glucosaminidase, amidase and peptidase
activities (25).
It is also known that certain antibiotics, mainly
of the penicillin and cephalosporin series, are
capable of killing microorganisms, presumably
by activating their autolytic wall enzymes (27).
These intraceUular enzymes are thought to be
controlled by endogenous lipid material (e.g.-
phospholipids, lipoteichoic acid) (27). Thus,
any agent present in leukocytes or in tissue
fluids, which will disrupt the balance between
autolytic enzymes and their naturally occurring
inhibitors, may lead to the activation of autolysins,
and concomitantly to the release of toxic
bacterial agents.
In view of the complex interrelationship which
exist between bacteria and host factors in infectious
and imflammatory sites, it was of
interest to clarify some of the mechanisms and
the factors involved in the biodegradation and
persistence of microbial constituents in tissues.
The following is an overview of our studies on
this subject, employing staphylococci and
streptococci as model systems, and using biochemical
and electron microscopical techniques
(28).
The peptidoglycan of Staphylococcus aureus
was labelled during the logarithmic phase of
growth with 14C-N-acetylglucosamine. When
such labelled cells were incubated for several
hours at 37 ~ in acetate buffer pH 5.0, with
small amounts of crude human leukocyte
extracts or with more purified lysosomal
extracts, a substantial amount of the radioactivity,
associated with the cell walls was
solubilized. Electron microscopical analysis of
these reaction mixtures revealed the accumulation
of cell wall fragments, and both amorphous
and intact cytoplasmic constituents still
retaining their typical morphologies (6, 7, 21,
22, 28, 29).
Since the pH optimum for this reaction process
was found to be on the acid side and compatible
with the pH optima of many of the acid hydrolases
known to be present in the leukocyte
preparation (1) we postulated that the breakdown
of the bacterial ceils was mediated by
acid hydrolases. The findings, however, that
heat treatment did not destroy the capacity of
the leukocyte extracts to induce cell wall degradation,
and that purified radiolabelled
staphylococcal cell walls became completely
refractory to the lytic effect of the extracts
(21, 22), suggested that the wall degradation
observed was probably not caused by the leukocyte
hydrolases. Since leukocyte lysosomes
are known to be rich in heat-stable argininerich
bactericidal cationic proteins (LCP) (30)
and in myeloperoxidase (MPO) (1) (also a
cationic protein) it was reasonable to try and
employ them, instead of the total leukocyte
mixture, to lyse the staphylococci. Indeed
we found that as little as 0.5-1/zg/ml of nuclear
histone, poly-L-lysine, poly-L-arginine or MPO
and 10-50/ag/ml of crystalline pancreatic
ribonuclease or cytochrome C (all cationic in
nature) were sufficient to induce massive loss
of cell wall material from 108 log-phase staphylococci.
Furthermore, small amounts of the
membrane.damaging agents phospholipase A2,
and polymyxins B and E were also capable of
inducing cell wall lysis, as determined by the
release of N-acetyl-glucosamine (8, 31-33).
Since purified staphylococcal cell walls (devoid
of cytoplasmic structures) were extremely
refractory to any of the cationic polyelectrolytes
or to the membrane-damaging agents, we
postulated that perturbation of the staphylococcal
membrane by these agents might have
resulted in the activation of endogenous enzymes
presumably associated with the autolytic
systems (21, 22, 25, 26). As autolytic wall
enzymes are known to be heat-labile (25, 26),
it was of in,terest to try and reactivate the lytic
process by the addition of freshly-harvested
viable staphylococci (as donors of autolysins)
to the heat killed radiolabeled staphylococci or to the purified labelled cell walls, in the
presence of several of the cationic proteins or
the membrane-damaging agents. Indeed, such
mixtures resulted in a substantial loss of radiolabelled
wall material. This process was completely
blocked by anionic polyelectrolytes.
We suggested, therefore, that the activators of
autolysins interacted with the viable bacteria
to release the autolytic enzymes, which in turn
attacked and degraded the radiolabelled substances.
Similar results were recently described
with Bacillus subtilis (34). It has also been
suggested that pneumococci, gonococci, meningococci,
Streptococcus faecalis and perhaps
listeriae may also likewise be degraded in vivo
following the activation of their autolysins, and
not through the direct action of lysosomal
enzymes (28).
Further experiments showed that Staphylococcus
aureus, which had been cultivated in the
presence of sub-inhibitory concentrations of
penicillin G, became much more susceptible to
wall lysis, following treatment with leukocyte
extracts (24,35) suggesting a collaboration
between/3-1actam antibiotics, leukocyte factors
and bacterial autolysins in bacteriolysis (see 27).
Other studies from our laboratory (23) have
also shown that contrary to the accepted belief,
both fresh and heat-treated human serum, when
properly diluted, also lysed log-phase grampositive
staphylococci and Streptococcus faecalis
at pH 5.0. Since anionic polyelectrolytes also
inhibited cell lysis induced by serum (23), and
since heat-killed bacteria became resistant to
lysis by serum, we postulated that, as in the
case of leukocyte extracts, lysis was induced by
a heat-stable factor, presumably ~-lysin of
platelet origin (4) present in serum, which
activated the autolytic systems of the bacteria.
To further elucidate the mechanism of bacteriolysis,
we have analyzed this process by electron
microscopy. In collaborative studies with Prof.
P. Giesbrecht and Dr. J. Wecke of the Robert
Koch Institute in Berlin, we found (36) that a
few hours after the addition of either crystalline
lysozyme (500/ag) or pancreatic ribonuclease
(50/ag/ml) to log-phase staphylococci, the
first signs of cell damage could already be seen.
These consisted of the formation of small,
periodically-arranged lytic sites between the
cell wall and the cytoplasmic membrane of the
cross wall. This was followed by the formation
of a distinct gap between the cell wall proper
and the cytoplasmic membrane. The degradation
of the peripheral cell wall continued to the
opposite side of the cell, and extended gaps
underneath the wall could be detected long
before the cell wall itself was peeled off as large
ribbons. The cross wall often appeared to be
already disintegrated during the early phase of
lysozyme or ribonuclease action. The release of
the wall left apparently intact protoplasts,
which still retained their original shape, and
even the invagination of the cross walls were
conserved. At this point over 70 \% of the toal
radioactivity associated with the cell wall was
solubilized after three to four hours, and the
radioactivity could not be sedimented at
100,000 {\textbullet} g suggesting the formation of solubilized
peptidoglycan. Since lysozyme which
had been heated to destroy its enzymatic
activity still retained its ability to induce cell
wall lysis, we postulated that lysozyme in this
system did not act as an enzyme but as a
cationic protein.
Further studies from our laboratory (28) and
in collaboration with the Robert Koch Institute
(to be published in detail) have revealed that
very similar ultrastructural changes in the
staphylococci also took place following phagocytosis
by mouse non-elicited macrophages in
culture. On the other hand, although staphylococci,
which had been injected into mouse or
rat tissues, and which were phagocytosed by
both PMNs and macrophages, underwent rapid
loss of their cytoplasmic constituents, presumably
by digestion with lysosomal hydrolases
(37-39), no apparent damageto the cell
walls was evident for many days, suggesting
that the autolytic wall enzymes might have
been inhibited.
Since the degradation of the staphylococcal
cell walls in vitro was completely inhibited by
a variety of anionic polyelectrolytes like
heparin, dextran sulfate, polyanethole sulfonate
(a synthetic heparin), as well as by cationic
polyelectrolytes like histones, poly-L-lysine,
poly-L-ar~inine, etc., when used at 10-100
/ag/ml/10 ~ staphylococci (concentrations 10-
100 fold higher than those employed to activate
the autolytic wall enzymes (see above),
we also postulated that a delicate balance
between activators and inhibitors may determine
whether or not bacterial wall material
may be degraded in tissue lesions in vivo
(40,41).
Finally, iecent studies (42) have also shown
that high-molecular-weight degradation products
of staphylococcal cell walls derived
following treatment of the bacteria either in
buffers (spontaneous wall lysis) or by small
amounts of leukocyte extracts, were found
to possess very strong chemot~ctic activities
for PMNs in vitro, and to induce severe inflammatory
lesions when injected intraarticularly
to rats.
Thus, it may be concluded that the fate of
bacterial peptidoglycans in leukocytes in
inflamed tissues may be dependent on the
one hand on the availability of agents capable
of activating autolytic wall enzymes in bacteria,
and on the other hand on the presence in tissues
of inhibitory substances (polyelectrolytes)
which are capable of blocking bacteriolysis. It
is, however, not aimed t9 rule out the possibility
that other still unknown mechanisms may
function in the complex milieu of inflammation,
which may bring about the biodegradation
of bacteria. The employment of certain
antibiotics and other pharmacological agents,
yet to be discovered, which will be capable of
changing the balance between activators and
inhibitors of autolytic enzymes, may contribute
to a better understanding of the mechanisms
involved in the survial and persistence
of microbial agents in vivo. It is also obvious
that the release of large quantities of microbial
constituents following incomplete biodegradation
may prove to be deleterious to
tissues.
Finally, our studies on staphylococci do not
shed light on the mechanisms of biodegradation
of other microbial species of medical imprtance,
and is only a reflection of one possible
mechanism, which may or may not be common
to all microorganisms.},
	author = {Isaac Ginsburg and Meir Lahav}
}
@article {1667,
	title = {How are bacterial cells degraded by leukocytes in vivo? An enigma},
	journal = {Clinical Immunology Newsletter},
	volume = {4},
	number = {11},
	year = {1983},
	pages = {147-153},
	abstract = {This year marks the centennial anniversary
of Elie Metchnikoff{\textquoteright}s discovery
of the pivotal role played by
"professional" phagocytes in body defenses
against invading microorganisms.
His cellular theory dealt with
the phagocytic events and the postphagocytic
killing, and also alluded to
the digestion of the internalized bacteria
by the "cystases," later shown to
be associated with the lysosomal apparatus
of leukocytes. Fo date, despite
the fact that numerous studies have described
in great detail the mechanisms
by which serum and leukocytes kill
microorganisms (1, 8, 13, 24), surprisingly
little is actually known about the
biochemical pathways of degradation
and mechanisms of disposal of microbial
constituents once they have been
sequestered within phagolysosomes (3,
9, 10, 13, 24). It is usually taken for
granted that the numerous hydrolytic
enzymes, including the key bacteriolytic
enzyme lysozyme (muramidase),
present in lysosomes of "professional"
phagocytic cells [granulocytes
or polymorphonuclear neutrophils
(PMNs), and macrophages] are capable,
(at least theoretically) of stripping
off bacterial coats, thus exposing
the peptidoglycan to cleavage by touramidase.
Yet, the majority of pathogenic
microorganisms are highly refractory
to lysozyme action (9, 10,
17). One should also bear in mind
that, while a massive breakdown of
microbial cell walls eventually may
lead to a bactericidal reaction, the
mere killing of a microorganisms, either
by leukocyte or by serum factors,
may not necessarily be followed by a
bacteriolytic reaction. The importance
of elucidating the mechanism of microbial
biodegradation in tissues stems
from the observation that in many infectious
diseases there is "storage" of
nonbiodegraded microbial cell wall
components within macrophages for
long periods, which may be responsible
for the perpetuation and propagation
of chronic inflammatory sequelae
and tissue destruction (10, 18).
We have recently postulated (11,
14, 15) that bacteriolysis, and the biochemical
degradation that ensues
after bacteria have been attacked by
serum or by leukocytes, may involve
close cooperation among heat-stable
serum factors, cationic proteins and
phospholipase A2 of leukocytes, and
heat-labile endogenous bacterial autolyric
wall enzymes. This cooperation
is affected markedly by anionic polyelectrolytes,
likely to accumulate in inflammatory
exudates, which may shut
down autolysis and, thus, contribute to
unfavorable postinfectious sequelae
(10, 19).
The present communication is a
summary of efforts from our laboratory
to gain insight into the mechanisms
of lysis of Staphylococcus attreus,
chosen as a model, by lysosomal
enzymes of human blood leukocytes
(3, 8-11, 14, 15, 19).},
	author = {Isaac Ginsburg and Meir Lahav}
}
