The success of
Staphylococcus aureus
as a human pathogen is influenced by its ability to elaborate factors
that prevent infection resolution by the host immune system. Such
immune-altering factors include complement inhibitory molecules,
antibody binding proteins, super-antigens, as well as potent cytolytic
peptides and pore-forming toxins. Here, we discuss one class of immune
cell-targeting toxins, the bi-component leukotoxins. These toxins are
believed to form octameric oligomers of alternating subunits on the
surface of host cells and insert β-barrel pores into cell membranes
leading to osmotic imbalance and cell lysis
[1].
We will discuss the reemerging interest in leukotoxins as potent
virulence factors with defined cellular targets, the implications of
their lethal and sublethal cellular effects, as well as challenges that
have restricted understanding of their functional activity in vivo,
while emphasizing areas of interest for future exploration. In addition,
we highlight studies supporting the development of antileukotoxin
antibodies and immunization strategies as potential modalities to
counter
S. aureus infection.
PVL and Beyond: A Reemerging Interest in Immune Cell-Targeting Toxins
The most studied of the leukotoxins produced by
S. aureus
is the Panton-Valentine Leukocidin (LukSF/PVL). Interest in this toxin
stems from its prevalence among current epidemic strains of
community-acquired methicillin-resistant
S. aureus (CA-MRSA)
[2].
Indeed, epidemiological evidence exists to link PVL to a number of
diseases including skin and soft tissue infections as well as
necrotizing pneumonia, for which CA-MRSA is so notorious
[2]–
[6].
Experimentally, assessment of the contribution of PVL to pathogenesis
has been plagued by conflicting results, owing to the apparent species
specificity of toxin action
[7].
Despite these experimental difficulties, PVL has been linked to both
necrotizing pneumonia and soft tissue infections using rabbit infection
models, although its actual contribution to skin and soft tissue
infection remains controversial
[8]–
[10].
However, PVL is only one of a family of four additional leukotoxins
present in strains causing human disease. These include the gamma
hemolysins (HlgAB and HlgCB), leukocidin ED (LukED), and leukocidin AB
(LukAB, otherwise known as LukHG
[11],
[12]) (
Figure 1).
A resurgence of interest in these leukotoxins, which are conserved in a
greater percentage of clinical isolates, has led to the discovery of
potential distinct roles for each toxin in
S. aureus pathogenesis.
Though
identified ~10 years ago, LukED had only been evaluated in terms of its
in vitro capacity to lyse human and rabbit neutrophils as well as red
blood cells (
Figure 1)
[13],
[14]. Recently however, LukED has been found to contribute to
S. aureus pathogenesis upon murine systemic infection due, in part, to toxin killing of phagocytic leukocytes in vivo
[15].
The newly identified leukotoxin LukAB was also shown to contribute to
distal tissue colonization upon infection with sublethal doses of MRSA
[12]. Additionally, among earlier reports of a role for Hlg in septic arthritis and endophthalmitis
[16],
[17], recent evidence from Malachowa and colleagues suggests a role for this toxin in bloodstream infection
[18]. Together, these data indicate leukotoxins likely contribute to multiple
S. aureus disease states in vivo. Future investigation into the contribution of each leukotoxin to
S. aureus
pathogenesis using multiple infection conditions and animal models will
serve to delineate each toxin's capacity to promote disease.
Challenging the Proposition of Strict Functional Redundancy
S. aureus leukotoxins exhibit lytic activity on host neutrophils, although some have greater perceived potency than others (
Figure 1).
Early studies gave considerable attention to this apparent redundancy
in toxin targeting using primary human and rabbit neutrophils
[19],
[20].
However, recent efforts are now moving toward investigation of the full
repertoire of cells killed by each leukotoxin, with the premise that
each may be unique in both the breadth and specificity of its cellular
targets despite significant similarities at the amino acid and
structural level. Studies by Holzinger et al. confirmed earlier work
describing the lytic capacity of PVL on neutrophils and monocytes, but
not lymphocytes
[21],
[22]. In addition, they demonstrated the lytic capacity of PVL on macrophages (
Figure 1)
[21]. HlgCB is toxic toward neutrophils and macrophages but also exhibits lytic activity on red blood cells
[23]. HlgAB, on the other hand, is nontoxic toward human macrophages but is potent on murine macrophages (
Figure 1)
[23]. LukAB is toxic toward neutrophils, monocytes, macrophages, and dendritic cells, but not the T cell line Jurkat
[12]. LukED is active against human and rabbit neutrophils, rabbit red blood cells, as well as murine leukocytes (
Figure 1)
[13]–
[15].
The subtle differences in leukotoxin activity on specific cell types
imply cellular recognition via unique factors. Thus, inferring direct
relationships between the potency of one leukotoxin and another is
challenging, as abundance or accessibility of specific cellular targets
may vary significantly on host cell surfaces. Deciphering the reasons
for varied potencies of the leukotoxins on similar cell types as well as
their mechanisms of cellular targeting will prove valuable in future
attempts to equate leukotoxin function with pathogenic outcomes.
The Lytic Versus Sublytic Hypothesis
It was recently demonstrated that the killing of host phagocytes during systemic infection of mice with
S. aureus is dependent on LukED production
[15]. Thus, the lytic capacity of LukED is likely a biologically relevant process during
S. aureus pathogenesis. Other studies with PVL demonstrate that leukotoxins may also elicit cellular effects at sublytic concentrations (
Figure 1)
[21],
[23]–
[28]. Notably, PVL induces inflammasome activation of both monocytes and primary macrophages at sublethal doses
[23].
Inflammasome activation in this context is believed to contribute to
the inflammatory response and subsequent neutrophil recruitment during
necrotizing pneumonia (
Figure 1)
[23]. Low concentrations of PVL also appear to prime PMNs for increased bacterial killing by promoting neutrophil activation (
Figure 1)
[24].
Unfortunately, investigation of the influence of sublytic toxin
concentrations on leukocytes in vivo has only been studied using PVL in
murine models
[29],
[30].
Such studies are limited due to an inability of PVL to lyse murine
cells. Thus, while the sublytic effects of PVL on leukocytes are
intriguing, the in vivo consequences of such effects in the presence of
active toxin are not understood. Lending credence to the hypothesis that
S. aureus toxins influence cellular signaling during
infection, the prototypical pore-forming cytotoxin alpha hemolysin
targets macrophages to induce inflammasome activation in vivo
[31],
[32].
It is possible that within a host both lytic and sublytic
concentrations of the bicomponent leukotoxins are also encountered
depending on the site and context of infection. Studies using active
toxins amenable to small animal models (such as LukED) may prove
valuable in determining the consequences of such sublytic effects.
Overcoming Species Specificity to Investigate Leukotoxin Function in Vivo
As
mentioned, studies of PVL function in vivo have been complicated by the
species specificity associated with cellular targeting. PVL has
negligible lytic activity on murine neutrophils but is potent on human
and rabbit cells
[33].
Similar studies have demonstrated poor lytic activity of LukAB on
murine and rabbit neutrophils, but potent activity on human neutrophils
[12],
[25]. Interestingly, LukAB still influences the pathogenesis of MRSA in murine systemic infection models
[12].
Future work aimed at deciphering the mechanism by which LukAB
facilitates pathogenesis in murine models will allow a better assessment
of this toxin's activities in vivo. Additionally, HlgCB kills human
macrophages but exhibits little lytic activity on murine macrophages
[23].
Thus, studies using murine models to evaluate these particular toxins
in vivo are best interpreted in light of their nonlytic effects. For
this reason, many PVL studies are now conducted using rabbit models of
infection
[8],
[9].
In contrast, LukED is toxic toward murine, rabbit, and human leukocytes
and is thus amenable to in vivo studies using murine models of
infection
[15]. Indeed, the lytic activity of LukED was recapitulated on phagocytic leukocytes in vivo
[15].
Thus, future studies of LukED are likely to provide a robust model of
leukotoxin function in vivo. The additional finding that HlgAB is lytic
on murine macrophages supports assessment of this toxin using murine
models
[23].
The Leukotoxins as Valuable Vaccine and Therapeutic Targets
Evidence indicating a role for each of the leukotoxins in the greater virulence of
S. aureus
implies potential value in targeting these molecules to counter
infection. Leventie et al. have generated humanized heavy-chain-only
antibodies and diabodies against PVL that show promise in their ability
to neutralize the damaging effects of the toxin in vivo
[34]. These same anti-PVL antibodies also block the activity of HlgCB on host cells
[34].
Dual neutralization by these antibodies is perhaps not surprising due
to the high degree of sequence similarity among leukotoxins but serves
as proof that it is possible to generate single antibodies or a subset
of antibodies with the ability to neutralize multiple leukotoxins,
thereby blunting disease progression
[35]. Vaccination of mice with PVL has likewise demonstrated promise toward reducing the pathogenic outcome of
S. aureus infection
[36],
though other studies, which passively immunized mice with serum from
PVL-immunized rabbits, lead to increased virulence for some strains
[30].
In either case, these murine studies should be interpreted with caution
given the low level of PVL lytic activity on murine cells. Even so,
both the humanized antibody and early stage vaccination studies suggest
that use of leukotoxins as immunizing agents is a potentially reasonable
approach toward promoting natural clearance of infection. Blocking
leukotoxin activity will necessitate targeting multiple toxins, as not
all strains contain the same leukotoxin profile and each toxin appears
to contribute variably to different disease states. A better
understanding of leukotoxin mode(s) of action in vivo, inherent
redundancies or lack thereof, and the intricacies of cellular targeting
will prove beneficial in our ability to initiate the development of
novel strategies to counter
S. aureus infection.
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