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. Author manuscript; available in PMC: 2012 Mar 25.
Published in final edited form as: Mol Microbiol. 2010 Dec 13;79(3):814–825. doi: 10.1111/j.1365-2958.2010.07490.x

Characterization of a new cytotoxin that contributes to Staphylococcus aureus pathogenesis

Ashley L DuMont 1, Tyler K Nygaard 2, Robert L Watkins 2, Amanda Smith 1, Lina Kozhaya 1, Barry N Kreiswirth 3, Bo Shopsin 4, Derya Unutmaz 1,4,5, Jovanka M Voyich 2, Victor J Torres 1,*
PMCID: PMC3312031  NIHMSID: NIHMS263241  PMID: 21255120

SUMMARY

Staphylococcus aureus is an important pathogen that continues to be a significant global health threat due to the prevalence of methicillin resistant S. aureus strains (MRSA). The pathogenesis of this organism is partly attributed to the production of a large repertoire of cytotoxins that target and kill innate immune cells, which provide the first line of defense against S. aureus infection. Here we demonstrate that leukocidin A/B (LukAB) is required and sufficient for the ability of S. aureus, including MRSA, to kill human neutrophils, macrophages and dendritic cells. LukAB targets the plasma membrane of host cells resulting in cellular swelling and subsequent cell death. We found that S. aureus lacking lukAB are severely impaired in their ability to kill phagocytes during bacteria-phagocyte interaction, which in turn renders the lukAB-negative staphylococci more susceptible to killing by neutrophils. Notably, we show that lukAB is expressed in vivo within abscesses in a murine infection model and that it contributes significantly to pathogenesis of MRSA in an animal hosts. Collectively, these results extend our understanding of how S. aureus avoids phagocyte-mediated clearance, and underscore LukAB as an important factor that contributes to staphylococcal pathogenesis.

Keywords: MRSA, Staphylococcus aureus, neutrophils, pathogenesis, leukotoxins

INTRODUCTION

Staphylococcus aureus is an extremely successful pathogen that poses a significant public health threat, and its treatment is complicated by the increasing prevalence of methicillin-resistant strains (MRSA) (Fridkin et al., 2005). The recent emergence of community-associated MRSA (CA-MRSA) infections of otherwise healthy individuals further highlights the virulence potential of this organism (Graves et al., 2010). S. aureus can infect a broad range of human tissues and organs resulting in potentially fatal diseases such as necrotizing fasciitis, pneumonia, endocarditis, sepsis, and toxic shock syndrome. A hallmark of staphylococcal infection is the formation of abscesses (Ogston, 1882), a battle ground where S. aureus combats leukocytes, the primary and most important line of defense against S. aureus infection (Lekstrom-Himes & Gallin, 2000, Verdrengh & Tarkowski, 1997, Gresham et al., 2000, Corbin et al., 2008). The pathogenesis of S. aureus depends partly on the production of an extensive repertoire of exoproteins and cell wall-anchored proteins that allow the organism to evade the innate immune system (Nizet, 2007, Foster, 2005, Graves et al., 2010).

The β-barrel family of pore-forming toxins is one group of staphylococcal cytotoxins that targets and kills mammalian cells. Among these toxins are the bi-component leukotoxins, which target polymorphonuclear cells (PMNs or neutrophils) (Menestrina et al., 2003). The bi-component leukotoxins are composed of a class S-subunit (slow eluted) and a class F-subunit (fast eluted), which act synergistically to form pores in the plasma membrane of target cells. The pores formed by these bi-component leukotoxins are typically cation selective and cell death seems to result from osmotic imbalance due to the flux of cations (Menestrina et al., 2003). Thus far, the repertoire of bi-component leukotoxins produced by S. aureus comprises γ-hemolysin (HlgAB and HlgCB), leukocidin E/D, leukocidin E/Dv (Morinaga et al., 2003), leukocidin M/F’, and Panton-Valentine leukocidin (PVL) also known as leukocidin S/F-PV (Menestrina et al., 2001). The focus of most studies in the staphylococcal toxin field has been on γ-hemolysin (Menestrina et al., 2003), PVL (Voyich et al., 2006, Labandeira-Rey et al., 2007, Loffler et al., 2010, Brown et al., 2009, Tseng et al., 2009, Varshney et al., 2010, Diep et al., 2010), and the β-barrel pore-forming toxin α-hemolysin (Hla) (Patel et al., 1987, Bubeck Wardenburg et al., 2007, Wardenburg & Schneewind, 2008). In addition to the bi-component leukotoxins, S. aureus also produces phenol soluble modulins (PSMs). These cytolytic peptides are associated with CA-MRSA virulence and have been shown to contribute significantly to human PMN lysis (Wang et al., 2007, Hongo et al., 2009). Although there has been a great deal of research dedicated to understanding the staphylococcal cytotoxins, the roles of each of these toxins is still unclear with regard to the ability of S. aureus to avoid phagocyte-mediated killing.

In this study, we characterize a new member of the staphylococcal bi-component leukotoxin family, which we have named leukocidin A/B (LukAB). Our data demonstrate that LukAB is predominantly responsible for the killing of human phagocytes through membrane disruption. LukAB plays an important role in the ability of S. aureus to target and kill neutrophils, protecting S. aureus from neutrophil-mediated killing. In addition, we found that LukAB contributes significantly to the pathogenesis of CA-MRSA in a vertebrate infection model. Thus, these data suggest that LukAB is an important staphylococcal toxin involved in the ability of S. aureus to avoid host defenses.

RESULTS

LukAB is a novel staphylococcal cytotoxin that targets and kills phagocytes

S. aureus strain Newman, a methicillin-sensitive S. aureus strain (MSSA), secretes a large number of proteins into the extracellular milieu (Torres et al., 2010). We observed that culture filtrates from this strain are cytotoxic towards human immune cell lines including Jurkat (T lymphocytes) and PMN-HL60 (neutrophil-like) cells (Fig. 1A-B). To elucidate the contribution of secreted toxins towards the S. aureus-mediated killing of human immune cells, we employed isogenic mutant strains lacking α-hemolysin (Δhla::bursa), γ-hemolysin (Δhlg::tet), or leukocidin ED (ΔlukED) (Fig. S1). The exoprotein profiles of these mutants are indistinguishable from that of the WT strain Newman indicating that the mutagenesis of these strains did not affect global production of secreted proteins (Fig. S2). Intoxication of Jurkat cells with culture filtrates from the isogenic mutants confirmed that α-hemolysin is predominately responsible for the observed decrease in cell viability (Fig. 1A) (Essmann et al., 2003). Surprisingly, intoxication of the PMN-HL60 cells with culture filtrate from the isogenic mutants revealed that Hla, HlgAB/CB, and LukED do not contribute to the killing of these cells (Fig. 1B).

Fig. 1. LukAB is a potent staphylococcal cytotoxin that kills human phagocytic cell lines.

Fig. 1

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A-B. Intoxication of Jurkat (A) or PMN-HL60 cells (B) with various dilutions of culture filtrate from the strain Newman (WT) and the indicated isogenic mutant strains. C. Abundance of toxins secreted by S. aureus strain Newman determined by LC-MS-MS and spectral counting. These data represent means of five independent experiments and the error bars represent the standard error of the mean (S.E.M.). D. Intoxication of PMN-HL60 cells with culture filtrate from WT containing an empty plasmid (WT/p), a strain lacking lukAB with an empty plasmid (ΔlukAB/p), and a strain lacking lukAB with a lukAB complementation plasmid (ΔlukAB/plukAB). E. Intoxication of PMN-HL60 cells with purified LukA (rLukA), LukB (rLukB), or a combination of rLukA and rLukB at the indicated concentrations. For the intoxications with both rLukA and rLukB, the total protein concentration is comprised of equal amounts of rLukA and rLukB, with each subunit at the indicated protein concentration. * indicates statistical significance from both rLukA and rLukB, P < 0.05. For panels (A, B and D), cell viability was monitored using CellTiter, where cells treated with medium were set at 100%. Results represent the average of triplicate samples ± standard deviation (S.D.). For panels (A and D), * indicates statistical significance from WT, ** indicates statistical significance from ΔlukAB/p, P < 0.05.

Analysis of the S. aureus strain Newman exoproteome revealed the presence of a bi-component leukotoxin-like protein (Fig. 1C), which we have denoted LukAB. The LukA and LukB polypeptides are encoded by the NWMN_1928 and NWMN_1927genes (Baba et al., 2008), which we have named lukA and lukB respectively. Aminoacid sequence alignment comparing LukA and LukB to staphylococcal leukocidin S-(LukE, LukEv, LukS-PV, LukS-I, HlgA, HlgC, LukM) and F-(LukD, LukDv, LukF-PV, LukF-I, HlgB, LukF’-PV) subunits revealed that both LukA and LukB group into new branches of a phylogenetic tree (Fig. S3). Levels of amino acid identity among previously known proteins within each subunit class are within 55% to 81%, whereas LukA only exhibits about 30% amino acid sequence identity with other S-subunits and LukB exhibits about 40% amino-acid sequence identity with other F-subunits.

To define the role of LukAB in S. aureus-mediated cytotoxicity, we generated an isogenic mutant strain lacking lukAB (ΔlukAB) which displays a normal exoprotein profile (Fig. S1-S2). Strikingly, we observed minimal death when PMN-HL60 cells were intoxicated with exoproteins from the lukAB-negative strain (Fig. 1D), even when the intoxication was extended to 24 hours (Fig. S4). The lack of cytotoxicity exhibited by the ΔlukAB strain was rescued by expressing lukAB in trans (Fig. 1D). To rule out the contribution of other factors present in the staphylococcal culture supernatant, we intoxicated PMN-HL60 cells with purified-recombinant LukA or LukB. Individual subunits exhibited no detectable cytotoxicity (Fig. 1E). In contrast, a combination of both subunits resulted in potent cytotoxicity in a dose-dependent manner (Fig. 1E).

LukAB targets the plasma membrane of host cells

Intoxication of PMN-HL60 cells with S. aureus exoproteins causes nuclei swelling, increased vacuolation and nuclear membrane separation, morphological changes absent from cells intoxicated with exoproteins from the lukAB-negative strain (Fig. 2A-B). The high-resolution electron microscopy (EM) images also revealed that LukAB disrupts the membrane of target cells, as we observed visible breaks in the plasma membranes of cells intoxicated with exoproteins from the WT strain, whereas the membranes of cells intoxicated with exoproteins from the lukAB-negative strain remained intact (Fig. 2C). Consistent with the EM data, cells intoxicated with exoproteins from the WT strain were permeable to SYTOX green, a cell-impermeable dye that binds DNA once inside cells, resulting in nuclear fluorescence. No difference was observed between the WT and the isogenic mutant strains lacking hla, hlgABC, and lukED in the cell permeability assay (Fig. 2D). In contrast, exoproteins from the isogenic lukAB-negative strain had little to no effect on cell permeability, a phenotype complemented when cells were intoxicated with exoproteins from the lukAB-negative strain provided with lukAB (Fig. 2D). Furthermore, a combination of purified-recombinant LukA and LukB, but not the individual toxins, caused membrane damage in a dose-dependent manner, indicating that LukAB disrupts the plasma membrane of target cells (Fig. 2E).

Fig. 2. LukAB disrupts the plasma membranes of target cells.

Fig. 2

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A. Light microscopy images of PMN-HL60 cells or PMN-HL60 cells intoxicated for two hours with 5% (v/v) culture filtrate from the indicated S. aureus strain Newman (WT or the ΔlukAB strain). B. EM images of PMN-HL60 cells incubated in medium alone or PMN-HL60 cells intoxicated for one hour with 2.5% (v/v) culture filtrate from indicated strains. Swollen cellular morphology is indicted with an arrowhead. C. High magnification EM images of the plasma membrane of a PMN-HL60 cell intoxicated as described in panel B. Membrane disruption is indicated with an arrowhead. D. Intoxication of PMN-HL60 cells with culture filtrate from the indicated strain Newman isogenic mutants and the ΔlukAB/plukAB strain. * indicates statistical significance from WT, ** indicates statistical significance from ΔlukAB/p, P < 0.05. E. Intoxication of PMN-HL60 cells with purified LukA (rLukA), LukB (rLukB), or a combination of rLukA and rLukB at the indicated concentrations as described in Fig. 1. * indicates statistical significance from both rLukA and rLukB, P < 0.05. For panels (D and E) cells with compromised membranes were stained with SYTOX Green and green-fluorescence intensity was measured. Results represent the average of triplicate samples ± S.D.

S. aureus kills primary human phagocytes in a LukAB dependent manner

We next sought to determine the contribution of LukAB to the ability of S. aureus to kill primary human cells. Intoxication of primary human monocytes, macrophages, and dendritic cells with culture filtrate from a WT strain or the isogenic cytotoxin mutant strains (Δhla::bursa, Δhlg::tet, ΔlukED, and ΔlukAB) revealed that LukAB is responsible for the cytotoxicity exhibited by S. aureus towards primary human phagocytes (Fig. 3A). LukAB also is responsible for the S. aureus mediated-killing of primary human PMNs (Fig. 3B), where cell death is a result of LukAB-mediated lysis as determined by measuring lactate dehydrogenase (LDH) release from PMNs (Fig. 3C). Exoproteins from the ΔlukAB strain cause minimal LDH release compared to exoproteins from the WT strain, a phenotype complemented by providing lukAB in trans (Fig. 3C). In addition, a combination of purified-recombinant LukA and LukB, but not the individual toxins, potently killed primary human PMNs (Fig. 3D).

Fig. 3. LukAB kills primary human phagocytes.

Fig. 3

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A. Intoxication of primary monocytes, macrophages, and dendritic cells (DC) with culture filtrate (2.5% v/v) from S. aureus strain Newman (WT) and the indicated isogenic mutant strains. Cell viability was monitored as described in Fig. 1. Results represent the mean from two donors, where cells from each donor were intoxicated with three independent exoprotein preparations, ± S.E.M. B. Intoxication of primary human PMNs with various dilutions of culture filtrates from the S. aureus strain Newman (WT) and the indicated isogenic mutant strains. Cell viability was monitored as described in Fig. 1. Results represent the mean from PMNs isolated from four donors ± S.E.M. C. Intoxication of primary human PMNs with culture filtrates (2.5% v/v) from the Newman WT/p, ΔlukAB/p, and ΔlukAB/plukAB strains. LDH-release was measured as an indicator of membrane disruption. Results represent the mean from PMNs isolated from six different donors ± S.E.M. D. Intoxication of primary human PMNs with purified rLukA, rLukB, or a combination of rLukA and rLukB at the indicated concentrations as described in Fig. 1. * indicates statistical significance from both rLukA and rLukB, P < 0.05. For panels (A-C) * indicates statistical significance from WT, ** indicates statistical significance from ΔlukAB/p, P < 0.05.

LukAB protects S. aureus from host-mediated killing

To rule out any potential artifact due to the use of culture supernatants or purified toxins, we examined the membrane integrity of PMN-HL60 cells infected with S. aureus WT and isogenic strains lacking hla, hlgACB, lukED, and lukAB. In these experiments membrane disruption is exclusively attributed to toxins that are actively secreted by and/or associated with the organism during co-culture with cells. As was demonstrated by the intoxication of cells with exoproteins from the isogenic mutant strains, these data revealed that LukAB is responsible for the membrane disruption caused by S. aureus and that Hla, Hlg and LukED do not contribute to the observed membrane damage (Fig. 4A). Similarly, LukAB is also responsible for disrupting the plasma membrane of primary human PMNs during co-culture with S. aureus (Fig. 4B).

Fig. 4. S. aureus lukAB mutants are attenuated in ex vivo infection models.

Fig. 4

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A. Infection of PMN-HL60 cells with the S. aureus strain Newman WT and the indicated isogenic mutants at various MOI. Results represent the average of triplicate samples ± S.D. B. Infection of primary human PMNs with the S. aureus strain Newman WT, the lukAB isogenic mutant strain and complemented strain (ΔlukAB/plukAB) at various MOI. Results represent the mean from PMNs isolated from five donors ± S.E.M. C. Viability of the indicated S. aureus strains upon human whole blood infection and upon infection of primary human PMNs. Colony forming units were normalized where input S. aureus strains were set at 100% viable. Results represent the mean from whole blood isolated from six donors or the mean from PMNs isolated from 12 donors ± S.E.M. For panels (A and B) mammalian cells with compromised membranes were stained with SYTOX Green as described in Fig. 2. For panels (A-C) * indicates statistical significance from WT, ** indicates statistical significance from ΔlukAB/p, P < 0.05.

To determine if LukAB can provide protection from phagocyte-mediated clearance, we measured the viability of the S. aureus WT and ΔlukAB mutant strains after ex vivo infection of human whole blood (Voyich et al., 2009). Disruption of lukAB impaired the ability of S. aureus to avoid whole blood-mediated killing, a phenotype rescued by providing the mutant strain with lukAB (Fig. 4C). We then investigated whether LukAB could also protect S. aureus from PMN-mediated killing. As with the whole blood assay, the lukAB mutant strain was also attenuated compared to the WT and complemented strains in the PMN infection model (Fig. 4C).

LukAB contributes to MRSA pathogenesis

To investigate if LukAB is produced by different staphylococcal strains, we generated polyclonal antibodies against LukA and LukB. We observed that LukA and LukB were produced by MSSA and MRSA strains (data not shown and Fig. 5A and Fig. S5). Of note, we were unable to detect LukAB in several clinical isolates (Fig. S5). We observed that LukB production was linked to the cytotoxic potential of the strain tested, since strains that produced high levels of LukB were significantly more cytotoxic than strains that produce low or undetectable levels of LukB (Fig. S5).

Fig. 5. LukAB contributes to the cytotoxicity of MRSA and is important for the pathogenesis of MRSA in vivo.

Fig. 5

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A. LukA, LukB, LukF-PV and α-toxin (Hla) production and the cytotoxic profiles of several MSSA and MRSA WT and isogenic ΔlukAB mutant strains against PMN-HL60 cells. PMN-HL60s were intoxicated with 10% (v/v) culture filtrate from the indicated strains and cell viability was monitored as described in Fig. 1. Results represent the average of triplicate samples ± S.D. B. Intoxication of primary human PMNs and primary human macrophages with culture filtrates (2.5% v/v) from the MRSA strain LAC, and the respective ΔlukAB isogenic mutant strains and complemented strains (ΔlukAB/plukAB). Cell viability was monitored as described in Fig. 1. Results represent the mean from PMNs and macrophages isolated from three to four donors ± S.E.M. C. Infection of primary human PMNs for one hour with the MRSA strain LAC WT, the lukAB isogenic mutant strain and complemented strain (ΔlukAB/plukAB) at various MOI. Results represent the mean from PMNs isolated from five donors ± S.E.M. D. Bioluminescent images of kidneys from mice infected with the WT S. aureus strain LAC containing pXen1 or the plukAB.Xen1. The kidneys of two representative mice per group are shown. E. Bacterial load recovered from the kidneys of mice infected retro-orbitally with the indicated S. aureus LAC strains. Each data point represents the number of bacteria (CFU) per milliliter of tissue homogenate in a single animal. Dashed line indicates the limit of detection. For panels (A-C and E) * indicates statistical significance from WT, ** indicates statistical significance from ΔlukAB/p, P < 0.05.

To explore the role of LukAB in the cytotoxic potential of MSSA and MRSA strains, we created several isogenic mutants lacking lukAB. Immunoblot analyses revealed that deletion of lukAB do not alter the production of PVL (LukF-PV) or Hla (Fig. 5A). Intoxication of PMN-HL60 cells with culture filtrates from these strains demonstrated that LukAB contributes to the cytotoxicity all of the strains tested, including the MSSA strain (BK4645) and PVL-producing MRSA strains (e.g. MW2, LAC, USA300-18807, USA300-18808, and USA300-18809), as the strains lacking lukAB were significantly less cytotoxic than the parental strains (Fig. 5A). As with strain Newman (Fig. 3), the CA-MRSA lukAB mutant strain LAC was also less cytotoxic to both primary human PMNs and macrophages compared to the WT and complemented strains (Fig. 5B). Infection of primary human PMNs with the LAC WT, ΔlukAB, and ΔlukAB-complemented strains demonstrated that the membrane disruption observed during the incubation of PMNs with LAC is due to LukAB (Fig. 5C).

To elucidate if lukAB is expressed during infection, we employed in vivo bioluminescence imaging (IVIS) in a S. aureus murine renal abscess model (Francis et al., 2000, Corbin et al., 2008, Torres et al., 2007). Animals were infected with the wild-type CA-MRSA strain LAC harboring either a promoterless control vector (pXen1; Xenogen), or the lukAB promoter fused to the modified luciferase operon of Photorhabdus luminescens (plukAB.Xen1). After 96 hrs, animals infected with the control strain containing the pXen1 vector exhibited no detectable luminescence in the kidneys (Fig. 5D). In contrast, luminescence was detected in the kidneys of animals infected with the lukAB reporter strain. Notably, the lukAB promoter was activated within abscesses, a site where S. aureus is in close contact with infiltrating neutrophils (Corbin et al., 2008, Torres et al., 2007).

The experiments described above suggest that LukAB plays a key role in the cytotoxicity of S. aureus strains. We next sought to investigate if LukAB contributes to S. aureus pathogenesis by employing a similar murine renal abscess model described above. Animals were infected with the CA-MRSA strain LAC WT, ΔlukAB and the complemented strain (ΔlukAB/plukAB). Enumeration of bacterial loads in the kidneys 96-hrs post infection revealed a 100-fold decrease in the number of mutant staphylococci as compared with WT, a phenotype restored by expression of lukAB in trans (Fig. 5E).

DISCUSSION

S. aureus remains to be a prominent pathogen on the global health scene due to the emergence of antibiotic resistant strains including HA-MRSA and CA-MRSA (Fridkin et al., 2005). The success of S. aureus as a pathogen is in part due to the elaboration of an arsenal of cytotoxins that allows the bacterium to evade host defenses by killing phagocytes and other immune cells (Menestrina et al., 2003, Nizet, 2007, Foster, 2005). In this study, we employed a series of isogenic mutants to systematically dissect the roles of individual cytotoxins produced by S. aureus in the staphylococcal mediated-killing of immune cells. This approach revealed that a new bi-component leukotoxin we have denoted LukAB, is predominantly responsible for death of human phagocytes caused by S. aureus strain Newman, a phenotype observed in both human cell lines and human primary cells. Our data demonstrate that LukAB targets phagocytes and disrupts the plasma membrane of these cells, which in turn leads to nuclear and cytoplasmic swelling and ultimately cell death.

Here we not only show that LukAB is important for the cytotoxic potential of strain Newman, but that LukAB also contributes to the cytotoxicity of MRSA strains, including the CA-MRSA strain LAC. While we were writing this manuscript, a recent study by Ventura et al. identified the cytotoxin LukGH by surveying the surface proteins of the CA-MRSA strain LAC (Ventura et al.). This study revealed that LukG and LukH are two of the most abundant surface proteins on strain LAC. By using culture supernatants from isogenic strains the authors found that LukGH and PVL act synergistically to form pores in PMN membranes. However, when cells were infected with the strain lacking lukGH it became apparent that LukGH but not PVL is responsible for the PMN lysis caused by strain LAC. Comparison of the LukGH with LukAB revealed that these toxins are the same, where LukA is LukH and LukB is LukG. In addition to the work by Ventura et al., this study demonstrates that LukAB also kills human macrophages, monocytes, and dendritic cells, and that this toxin confers resistance to S. aureus from primary human whole blood- and PMN-mediated killing. Furthermore, we demonstrate that the lukAB promoter is activated in vivo and that LukAB is required for the full pathogenesis of the CA-MRSA strain LAC in a murine model of infection.

Taken together, this study demonstrates that LukAB is a potent toxin involved in protecting S. aureus (MSSA and MRSA) from innate immune cells by eliminating phagocytes through membrane disruption. Our observation that LukAB also targets and kills human macrophages and dendritic cells, suggests that this toxin may also play a role in disrupting the priming of the adaptive immune response to S. aureus, which could further contribute to the enhanced pathogenesis of this bacterium.

EXPERIMENTAL PROCEDURES

Bacterial strains and culture

S. aureus isolate Newman (Duthie & Lorenz, 1952), the colonizing strain BK4645 (Shopsin et al., 2008), and the MRSA S. aureus pulse field gel electrophoresis types USA400 (strain MW2) (Baba et al., 2002), and USA300s (Kennedy et al., 2008) were used in all experiments as the wildtype strain (unless explicitly stated). S. aureus strains were grown on tryptic soy broth (TSB) solidified with 1.5% agar at 37°C or in TSB with shaking at 180 rpm, unless otherwise indicated. When appropriate, TSB was supplemented with chloramphenicol at a final concentration of 10 μg/ml, erythromycin 10 μg/ml, or tetracycline at 4 μg/ml.

Escherichia coli DH5α was used to propagate plasmids, ER2566 (New England BioLabs) and T7 (New England BioLabs) were used as expression strains. DH5α strains were grown in Luria Bertani (LB) agar and LB broth supplemented with 100 μg/ml of ampicillin. The expression strains were grown in Terrific Broth supplemented with 100 μg/ml of ampicillin or 25 μg/ml of kanamycin.

Mammalian cell lines

HL60 cells (ATCC CCL-240), a human promyelocytic cell line and Jurkat cells (TIB-152), a human T lymphocyte cell line, were grown in Roswell Park Memorial Institute 1640 medium (RPMI; Cellgro) supplemented with 10% of heat inactivated fetal bovine serum (FBS) at 37°C with 5% CO2. To differentiate HL60 cells into neutrophil-like cells (PMN-HL60), cultures were supplemented with 1.5% (v/v) dimethylsulfoxide (DMSO) and grown for 3-4 days.

Primary human monocytes, macrophages, and dendritic cells

Blood samples were obtained from anonymous healthy donors as buffy coats (New York Blood Center). New York Blood Center obtains written informed consent from all participants involved in the study. Monocytes (CD14+) cells were isolated from PBMC using anti-CD14 antibody coated bead based sorting (Miltenyi) and were typically >99% CD14+ pure. Monocyte-derived dendritic cells (DCs) were generated from CD14+ cells by supplementing the culture medium with human GMCSF (50 ng/ml) + IL-4 (40 ng/ml) (Manel et al., 2010). Monocyte-derived macrophages were generated from CD14+ cells by supplementing the culture medium with human GMCSF (50 ng/ml) (Gramberg et al., 2010). Cells were cultured for 4 days in the differentiation condition prior to use.

Generation of mutant strains and complementation plasmids

Isogenic mutants lacking the hla and hlgACB gene derived from S. aureus strain Newman were previously described (Bae et al., 2004, Supersac et al., 1998). The mutants lacking lukED (NWMN_1719 and NWMN_1718 respectively) (ΔlukED) and lukAB (NWMN_1928 and NWMN_1927 respectively) (ΔlukAB) were derived from S. aureus strain Newman and were constructed using the pKOR-1 plasmid as described previously (Bae & Schneewind, 2006). Briefly, sequences flanking the lukED locus were PCR amplified with primers V J T 1 0 4 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTTGAAGTTAAGGCCTAC-3′ ) a n d VJT102 (5′-TCCCCCCGGGTAAAGAAACTAATCCTGG-3′) for the upstream fragment and primers VJT71 (5′GGGGACCACTTTGTACAAGAAAGCTGGGTCATTTATTCGACTAGCAAC-3′) and VJT70 (5′-TCCCCCCGGGCAAATTTATTCTAGATTATTTC-3′) for a downstream fragment. The PCR amplicons were digested with Xma1 and assembled into pCR2.1 (Invitrogen). A PCR amplicon of the joined DNA fragment was recombined into pKOR1 resulting in the pKOR-1ΔlukED and pKOR-1ΔlukAB plasmids. Deletion of the lukED and lukAB loci was achieved by allelic replacement as described previously (Bae & Schneewind, 2006). Mutagenesis was confirmed by PCR (fig. S1).

The lukAB complementation plasmid was generated by PCR amplifying the lukAB locus including its endogenous promoter with primers VJT144 (5′-ccccGAATTCGTGTTATTTGATTTCGTTCTATG-3 ′ ) and VJT260 ( 5 ′-cccGTCGACTTATTTCTTTTCATTATCATTAAGTACTT). The PCR amplicons were digested with EcoR1 and Sal1 and cloned into a similarly digested pOS1 plasmid (Schneewind et al., 1992).

Exoprotein production

For the production of filtered culture supernatants (exoproteins), S. aureus strains were grown in RPMI 1640 (Invitrogen) supplemented with 1% casamino acids (RPMI+CAS) (Torres et al., 2010). When required, the culture medium was supplemented with 10 μg/ml of chloramphenicol, 10 μg/ml of erythromycin, or 4 μg/ml of tetracycline. Overnight cultures were grown in 5 ml in 15 ml tubes held at a 45° angle and incubated at 37°C with shaking at 180 rpm. Following overnight cultures, bacteria were subcultured at a 1:100 dilution and grown as described above for 5 hrs. Bacteria were then pelleted by centrifugation at 4000 rpm and 4°C for 15 min. Supernatants containing exoproteins were collected, filtered using a 0.2 μm filter, and stored at −80°C.

SDS-PAGE of secreted proteins

Exoproteins in S. aureus culture supernatants were precipitated with 10% trichloroacetic acid (TCA) (v/v). The precipitated proteins were washed once with 100% ethanol, air-dried, resuspended with 30 μl of SDS-Laemmli buffer and boiled at 95°C for 10 min. Proteins were separated using 15% SDS-PAGE gels and stained with Coomassie brilliant blue.

Precipitated exoproteins were separated on 10% SDS-PAGE gels then transferred to nitrocellulose membranes. The membranes were incubated with polyclonal antibodies against LukB, LukF-PVL (Yoong & Pier, 2010), and α-toxin (Menzies & Kernodle, 1996) which were detected with AlexaFluor-680-conjugated anti-rabbit secondary (Invitrogen) antibody diluted 1:25,000. Membranes were dried and scanned using an Odyssey infrared imaging system (LI-COR Biosciences).

Proteomic analysis of S. aureus strain Newman culture supernatants

Exoproteins were collected, processed, and shotgun proteomic was performed as described previously (Torres et al., 2010).

Generation of the anti-LukA and LukB polyclonal antibodies

The lukA and lukB genes were amplified from S. aureus DNA with Vent polymerase (New England Bio Labs ) using the following primers : VJT203 ( 5 ′-gggCATATGAATTCAGCTCATAAAGACTCTCAA and VJT204 (5′-cccGTCGACTCCTTCTTTATAAGGTTTATTGTC) for lukA and VJT209 (5′-gggCATATGAAGATTAATTCTGAAATCAAACAAG-3 ′ ) and VJT210 ( 5 ′-cccGTCGACTTTCTTTTCATTATCATTAAGTACTT-3′) for lukB. The lukA and lukB gene products were digested with Nde1 and Sal1 (New England BioLabs) and ligated into the pET41b vector (Novagen), fusing the coding sequence of a histidine-tag to the 3′-region of the genes. The constructs were first transformed into DH5α cells and then transformed into the E. coli expression strain ER2566 (New England BioLabs). The transformants were grown in Terrific Broth supplemented with kanamycin (25 μg/ml) for 2.5 hrs at 37°C and expression of lukA and lukB was induced with 0.3 mM β-D-1-thiogalactopyranoside (IPTG) at 37°C for 2 hours with 180 rpm shaking. The cells were pelleted and resuspended in 1× tris buffered saline (TBS) (50 mM Tris, 150 mM NaCl, pH 7.5) and sonicated on ice for 1min (10 sec pulses). The lysed bacteria were ultracentrifuged for 30 min at 50,000 rpm. In order to purify the C-terminal His6-LukA and LukB under denaturing conditions, the pellets were resuspended in lysis buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea, pH 8.0) and incubated at room temperature for 30 min on a rotisserie. The samples were centrifuged at 13,000 rpm for 30 min and the supernatants were applied to a column containing Ni-NTA resin (Qiagen). The column was washed two times with wash buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea, pH 6.3) and the proteins were eluted from the columns using elution buffer (100 mM NaH2PO4, 10 mM Tris, 8 M urea) at pH 5.9 and at pH 4.5. Purified His6-LukA amd LukB were quantified using the Thermo Scientific Pierce BCA Protein Assay Kit. The LukB polyclonal antibody was generated by Covance Inc. and the LukA polyclonal antibody was generated by Pacific Immunology Corp. by immunizing rabbits with the respective purified-denatured protein.

Purification of recombinant LukA and LukB

lukA and lukB were amplified from S. aureus strain Newman DNA using the following primers: VJT300 (5′-ccccCTCGAGAATTCAGCTCATAAAGACTCTCAAG-3 ′ ) a n d V J T 3 0 1 ( 5 ′-ccccGGATCCTTATCCTTCTTTATAAGGTTTATTGTC-3′) for lukA and VJT302 (5′-ccccCTCGAGAAGATTAATTCTGAAATCAAACAAG-3 ′ ) a n d V J T 3 0 3 (5′-ccccGGATCCTTATTTCTTTTCATTATCATTAAGTACTTT-3′) for lukB. The lukA and lukB gene products were digested with Xho1 and BamH1 (New England BioLabs) and ligated into the pET14b vector (Novagen) fusing the coding sequence of a histidine-tag to the 5′-region of lukA and lukB. The expression plasmids were transformed into the E. coli strain DH5α and the plasmid fidelity was confirmed through sequencing. The plasmids were purified and transformed into the expression E. coli strain T7. The transformants were grown in Terrific Broth with 100 μg/ml of ampicillin at 37°C until cultures reached an A600 of ~0.5. The expression of 6-his-tagged LukA or 6-hisLukB was induced with 0.4 mM IPTG at 37°C, for 3hrs, with 180 rpm shaking. After the induction, the cells were harvested through centrifugation at 4000 rpm at 4°C for 15min and then resuspended in 1× TBS (50 mM Tris, 150 mM NaCl, pH 7.5). The bacterial cells were sonicated on ice for 2 min (10 sec pulses). The lysed bacteria were ultracentrifuged for 30 min at 50,000 rpm. Recombinant LukA and LukB (rLukA and rLukB) were purified under denaturing conditions using a Ni-NTA column as described above. The fractions containing purified protein, as determined by SDS-PAGE, were pooled, diluted 1:1 in 1X TBS, and dialyzed in 1X TBS at 4°C overnight to remove the urea and allow refolding. Purified 6-his-tagged LukA and LukB were quantified using the Thermo Scientific Pierce BCA Protein Assay Kit.

Cytotoxicity assays

To evaluate the viability of mammalian cells after intoxication with S. aureus leukocidins, cells were plated in 96-well flat-bottom tissue culture treated plates (Corning) at 1×105/well in a final volume of 100 μl of RPMI (Cellgro) supplemented with 10% of heat inactivated FBS. Cells were intoxicated for 2hrs at 37°C, 5% CO2 with serial 2-fold dilutions of culture filtrate from three independent S. aureus colonies ranging from 20% to 0.16% (v/v) or cells were intoxicated for 1 hour with rLukA (2.9-0.4 μM), rLukB (2.9-0.4 μM), or a mixture of rLukA+rLukB (1.5-0.2 μM final concentration). Controls for 100% viability included 20% (v/v) tissue culture media (RPMI+10% heat-inactivated FBS), and 20% v/v S. aureus growth medium (RPMI+Cas). 0.1% v/v TritonX100 was used as a control for 100% cell death. After the intoxication, 10 μl of CellTiter (Promega) was added to each well and the cells were incubated for an additional 2 hours at 37°C, 5% CO2. CellTiter monitors metabolically active cells (indicated by a color change), a property that is lost in dead cells. The colorimetric reaction was measured at 492 nm using a Perkin Elmer Envision 2103 Multilabel Reader. Percent viable cells were calculated using the following equation: % Viability = 100 × [(Ab492Sample − Ab492TritonX) / (Ab492Tissue culture media)].

Transmission Electron Microscopy (TEM)

PMN-HL60 and THP-1 cells were intoxicated with exoproteins (5-2.5% v/v) from WT S. aureus Newman and a strain lacking lukAB for 1 hour at 37°C with 5% CO2. Cells were then fixed in 0.1 M sodium cacodylate buffer (pH 7.2), containing 2.5% glutaraldehyde and 2% paraformaldehyde for 2 hours and post-fixed with 1% osmium tetroxide for 1.5 hours at room temperature, and en bloc stained with 1% uranyl acetate. The cells were dehydrated in ethanol and embedded in EMbed 812 (Electron Microscopy Sciences, Hatfield, PA). Semi-thin sections were cut at 1 μm and stained with 1% Toluidine Blue to evaluate the quality of preservation. Ultrathin sections (50 nm) were post stained with uranyl acetate and lead citrate and examined using Philips CM-12 electron microscope (FEI; Eindhoven, The Netherlands) and photographed with a Gatan (4k x2.7k) digital camera (Gatan, Inc., Pleasanton, CA).

Membrane damage assay

To evaluate the integrity of host cell plasma membranes, we employed SYTOX green (Invitrogen). Healthy cells are impermeable to SYTOX green, but become permeable to the dye once the cell membrane integrity has been compromised. Inside the cells, SYTOX green binds to DNA exhibiting strong fluorescence. To evaluate the integrity of host cell membranes after intoxication, cells were intoxicated as described above. After the intoxication, cells were transferred to a tissue culture treated 96-well v-bottom plate (Corning) and centrifuged at 1500 rpm for 5min. The supernatant was discarded and the pellets were resuspended in 100 μl of PBS + SYTOX green (0.1 μM; Invitrogen). The cells were then transferred to a 96-well clear-bottom black tissue culture treated plate (Corning) and incubated at room temperature in the dark for 10 min. Fluorescence was measured using a Perkin Elmer Envision 2103 Multilabel Reader (Excitation 485 nm, Emission 535 nm).

Ex vivo infection assays

S. aureus was cultured as described above. The bacteria were pelleted and washed with 1× PBS and resuspended in 5 ml of 1× PBS. 1×105 mammalian cell/well in a final volume of 100 μl of RPMI supplemented with 10% of heat inactivated FBS were infected with a MOI of 100, 50, 10, or 1 of the S. aureus strains in 96-well flat-bottom tissue culture treated plates at 37°C, 5% CO2 for 2 hrs. Membrane disruption was evaluated using the membrane damage assay described above. MOI were determined by serially diluting the input cultures and counting CFUs on tryptic soy agar (TSA) plates.

Human neutrophil assays PMNs

PMNs were isolated from venous blood and killing of S. aureus by human PMNs was determined as described previously (Voyich et al., 2005, Voyich et al., 2006). Lysis of PMNs by S. aureus was determined by the release of lactate dehydrogenase (LDH), using the Cytotoxicity Detection Kit (Roche Applied Sciences) as described previously (Voyich et al., 2005, Voyich et al., 2006). PMN viability and membrane damage were also monitored with Cell Titer (Promega) and SYTOX green, respectively, as described above.

Murine model of infection

All procedures involving animals were approved by NYU School of Medicine Institutional Animal Care and Use Committee. All animal experiments were performed in accordance to NIH guidelines, the Animal Welfare Act, and US federal law.

In vivo bioluminescence imaging (IVIS). Seven-week-old Swiss Webster (Hsd: ND4) female mice (Harlan Laboratories) were infected retro-orbitally with approximately 1×107 CFU of S. aureus strain LAC harboring either a promoterless control vector (pXen1), or the lukAB promoter fused to the modified luciferase operon of Photorhabdus luminescens (plukAB.Xen1). Four days post-infection, mice were euthanized with CO2 and kidneys were removed and imaged using an IVIS XR instrument (Caliper LifeSciences).

For the enumeration of bacterial burden, Hsd: ND4 mice were infected with 1×106 CFU of LAC strains as described above. Four days post-infection, mice were euthanized with CO2, kidneys were removed, homogenized in sterile PBS, serially diluted and plated on TSA for colony forming unit (CFU) counts. Two independent experiments were performed with 10 mice per strain.

Statistics

Data were analyzed using a one-way ANOVA and Tukey’s posttest (GraphPad Prism version 5.0; GraphPad Software) unless indicated otherwise. Data presented here are from one of at least three independent experiments that gave similar results.

Supplementary Material

Supp Figure S1-S5

ACKNOWLEDGEMENTS

We thank members of the Torres laboratory, and Drs. Martin Blaser, Timothy L. Cover, Heran Darwin, and Eric P. Skaar for critical reading of this manuscript. We also thank Mark DeWald and Alka Khaitan for help during the PMN preparation, Meredith Benson for help with the IVIS studies, Jennifer Philips for advice with SYTOX green assays, Alice Liang and Eric W. Roth (Image core facility, New York University School of Medicine) for performing the Electron Microscopy experiments, and W. Hayes McDonald (The Mass Spectrometry Research Center, Vanderbilt University Medical Center) for performing the shotgun proteomics analyses. We are also grateful to Drs. Eric Skaar for the generous gift of S. aureus strain Newman, hla::bursa mutant, Timothy Foster for the gift of the hlgACB::tet mutant S. aureus strain, Douglas Kernodle for providing the α-toxin antibody, and Jerry Pier for providing the anti-PVL antibodies. This research was enabled by New York University School of Medicine Development Funds (V.J.T), an American Heart Association Scientist Development Grant (09SDG2060036) (V.J.T), and by NIH-PAR98-072 and NIH-NCR (J.M.V.), and NIH Training Grant 5T32 AI007180-27 (A.L.D.).

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Supplementary Materials

Supp Figure S1-S5
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