Sword and shield: Linked group B streptococcal ␤-hemolysin͞cytolysin and carotenoid pigment function to subvert host phagocyte defense George Y. Liu*, Kelly S. Doran*, Toby Lawrence†, Nicole Turkson‡, Manuela Puliti§, Luciana Tissi§, and Victor Nizet*‡¶ Departments of *Pediatrics and †Pharmacology and ‡Center for Marine Biotechnology and Biomedicine, The Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093; and §Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, 06122 Perugia, Italy Communicated by Donald R. Helinski, University of California at San Diego, La Jolla, CA, August 23, 2004 (received for review January 12, 2004) Group B Streptococcus (GBS) is a major cause of pneumonia, A clinical association with invasive human infections implies bacteremia, and meningitis in neonates and has been found to persist that GBS can sometimes survive innate host defense mecha- inside host phagocytic cells. The pore-forming GBS ␤-hemolysin͞ nisms called upon to clear the bacteria from the bloodstream and cytolysin (␤H͞C) encoded by cylE is an important virulence factor tissues. A principal role in innate immunity is played by host as demonstrated in several in vivo models. Interestingly, cylE neutrophils and macrophages, which can engulf and kill bacteria deletion results not only in the loss of ␤H͞C activity, but also in the by generation of reactive oxygen species and other antimicrobial loss of a carotenoid pigment of unknown function. In this study, substances within the phagolysosome. Although streptococci are we sought to define the mechanism(s) by which cylE may contrib- commonly thought of as ‘‘extracellular pathogens,’’ GBS can ute to GBS phagocyte resistance and increased virulence potential. survive for prolonged periods within the phagolysosome of We found that cylE-deficient GBS was more readily cleared from a macrophages (8, 9). And, although GBS lack the neutralizing mouse’s bloodstream, human whole blood, and isolated macro- enzyme catalase, they can be Ͼ10 times more resistant to killing phage and neutrophil cultures. Survival was linked to the ability of by H2O2 than catalase-positive Staphylococcus aureus (10). The ␤H͞C to induce cytolysis and apoptosis of the phagocytes. At a mechanisms responsible for the enhanced survival of GBS are lower bacterial inoculum, cylE also contributed to enhanced sur- unknown. vival within phagocytes that was attributed to the ability of Previous interpretations of the role played by the GBS ␤H͞C carotenoid to shield GBS from oxidative damage. In oxidant killing in animal virulence have focused on cytolytic injury to host cells assays, cylE mutants were shown to be more susceptible to or stimulation of inflammatory responses. Here we ask the hydrogen peroxide, hypochlorite, superoxide, and singlet oxygen. question of whether the decreased virulence of GBS cylE Together, these data suggest a mechanism by which the linked mutants could reflect an important role of this gene in protecting cylE-encoded phenotypes, ␤H͞C (sword) and carotenoid (shield), GBS against host phagocytic clearance. We provide evidence act in partnership to thwart the immune phagocytic defenses. that cylE contributes to GBS survival in neutrophils and mac- rophages and show that this effect is due to not only toxic ␤ ͞ roup B Streptococcus (GBS) is the leading cause of invasive properties of H C but also protection by the linked carotenoid Gbacterial infections in human newborns and is increasingly pigment against oxidative burst killing mechanisms. recognized as a pathogen in adult populations, including the Materials and Methods elderly, pregnant women, and diabetics. One important viru- ͞ lence factor of GBS is a surface-associated toxin known as the Bacteria and Cell Lines. GBS used were WT strains NCTC10 84 ϭ ␤-hemolysin͞cytolysin (␤H͞C). ␤H͞C is responsible for the (serotype V, hemolytic titer 64 units) and A909 (serotype Ia, ϭ characteristic zone of clearing around GBS colonies grown on hemolytic titer 4 units) and the corresponding nonhemolytic, ⌬ blood agar plates and is capable of forming pores in a variety of nonpigmented isogenic allelic exchange mutants NCTC:cylE cat ␤ ͞ and A909:cylE⌬cat (3, 5). Here we use the abbreviated designations eukaryotic cell membranes. H C is thought to contribute to ⌬ ⌬ GBS pathogenicity by virtue of its cytolytic properties, its ability V-wt, Ia-wt, V cylE, and Ia cylE, respectively. Bacteria were to promote bacterial invasion of epithelial and endothelial grown in Todd–Hewitt broth (THB) or on Todd–Hewitt agar barriers, and its activation of host cytokines and other inflam- (THA) (Difco). J774 (a murine macrophage-like cell line), primary matory mediators. GBS ␤H͞C mutants exhibit decreased viru- murine macrophages, and human neutrophils were maintained in lence in animal models of sepsis and meningitis (1, 2). GBS RPMI medium 1640 plus 10% FBS. ␤H͞C-mediated cytotoxicity is blocked by the surfactant phos- pholipid dipalmitoyl phosphatidylcholine (DPPC), perhaps ex- Murine Model of Sepsis. Six- to 8-week-old CD-1 mice (Charles 8 plaining in part the increased susceptibility of premature, River Laboratories) were injected in the tail vein with 10 early surfactant-deficient neonates to severe GBS pneumonia (3). logarithmic-phase GBS and monitored daily for survival. At 24 h, Transposon mutagenesis studies mapped GBS ␤H͞C activity bacteremia was assessed by blood collection and enumeration of to the cyl operon (4). A single ORF, cylE, is both necessary for GBS colony-forming units (cfu) on THA. GBS hemolysin production and sufficient to confer ␤-hemolysis to Escherichia coli (5). The predicted 79-kDa protein product, Human Whole Blood Killing Assay. GBS was grown to early logarith- mic phase, washed, and resuspended in PBS. Inocula of 104 cfu in CylE, does not share homology with other proteins in the ␮ ␮ GenBank databases. Interestingly, the cylE gene is also required 100 l were mixed with 300 l of freshly drawn human blood in for GBS production of an orange carotenoid pigment, a unique ␤ feature that is useful in distinguishing GBS from other -hemo- Abbreviations: ␤H͞C, ␤-hemolysin͞cytolysin; cfu, colony-forming units; DPI, diphenylene lytic streptococci (6). GBS mutants in which the cylE gene is iodonium; DPPC, dipalmitoyl phosphatidylcholine; GBS, group B Streptococcus; moi, mul- removed are invariably both nonhemolytic and nonpigmented, tiplicity of infection; THA, Todd–Hewitt agar; THB, Todd–Hewitt broth; TUNEL, terminal and both phenotypes are restored in single-gene complementa- deoxynucleotidyltransferase-mediated dUTP nick end labeling. ¶ tion experiments where cylE is returned on a plasmid vector To whom correspondence should be addressed. E-mail: [email protected]. MICROBIOLOGY (4, 5, 7). © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0406143101 PNAS ͉ October 5, 2004 ͉ vol. 101 ͉ no. 40 ͉ 14491–14496 Downloaded by guest on October 2, 2021 heparinized tubes, and incubated for3hwithagitationat37°C, and medium 1640 and resuspended in RPMI medium 1640 plus 10% dilutions were plated on THA for enumeration of cfu. FBS plus 5 ␮g͞ml penicillin and 10 ␮g͞ml gentamicin to kill extracellular GBS. At specified time points thereafter, the cells Neutrophil Purification. Human neutrophils were purified from were washed two times with PBS and lysed with 0.02% Triton healthy human volunteers by using a Histopaque gradient X-100, and dilutions were plated on THA to count intracellular (Sigma) according to the manufacturer’s directions. Neutrophil bacterial cfu. To assess oxidative burst function, supernatant was purity was Ͼ95%. aspirated and replaced with 0.5 mg͞ml nitroblue tetrazolium (NBT) solution (Sigma). After 30 min at 37°C, the supernatant Macrophage and Neutrophil Killing Assays. Three days after i.p. was removed, and adherent cells were washed and extracted with ͞ injection of 3 ml of sterile thioglycolate broth, C57BL 6 mouse 200 ␮l of DMSO and assayed colorimetrically at 580 nm. (Charles River Laboratories) macrophages were isolated by peri- toneal lavage with PBS. Specific inocula of logarithmic-phase GBS Oxidant Killing Assays. Early stationary-phase GBS was spun down were added and brought into proximity with human neutrophils or and resuspended in PBS or fresh THB. H O was added at a final ϫ 2 2 murine macrophages by centrifugation at 600 g for10min.After concentration of 0.03% and GBS was incubated at 37°Cfor2h, 3–4hat37°Cin5%CO2, Triton X-100 (0.02% final) was added to at which time 1,000 units of catalase was added to quench lyse the phagocytes. Lysates were plated on THA at 37°Cfor ␤ ͞ remaining H2O2. Bacteria were plated on THA medium to enumeration of cfu. In H C-blocking experiments, the inhibitor calculate cfu. For the singlet oxygen assay, early stationary-phase DPPC was suspended in PBS by sonication and added to GBS for GBS was incubated in a 24-well culture plate at 37°Cinthe 5 min before incubation with macrophages. presence or absence of 0.01–0.2 mg͞ml methylene blue and 10 cm from a 100-watt light source. Control plates were handled Macrophage Cytotoxicity and Apoptosis Assays. To determine cy- identically but wrapped in foil. Bacterial viability was assessed totoxicity, macrophages were washed free of GBS by using PBS, after 3–5 h by plating on THA. Paraquat (30 mM) and sodium incubated in 0.4% trypan blue for 30 min at 37°C, fixed in 4% hypochlorite (0.2%, Sigma) were used as sources of superoxide paraformaldehyde, and counterstained in 0.1% eosin. For DNA and hypochlorite in similar assays. For rescue studies, bacteria fragmentation studies, logarithmic-phase GBS was added to 4 ϫ were mixed in THA supplemented with 1͞10th to 1͞80th vol of 106 macrophages and centrifuged as before. After2hat37°C, the cells were washed twice and resuspended in fresh RPMI medium 1640 plus 10% FBS plus 10 ␮g͞ml gentamicin plus 5 ␮g͞ml penicillin. At 24 h, the phagocytes were spun down, lysed in a solution containing 50 mM Tris (pH 7.2), 10 mM EDTA, 0.01% SDS, 0.2 mg͞ml RNase, and 0.5 mg͞ml proteinase K (4 h at 55°C), and DNA was harvested by ethanol precipitation for laddering analysis on a 2% agarose gel.