Emergence of a Bacterial Clone with Enhanced Virulence by Acquisition of a Phage Encoding a Secreted Phospholipase A2 Izabela Sitkiewicz*, Michal J

Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2 Izabela Sitkiewicz*, Michal J. Nagiec*†, Paul Sumby*, Stephanie D. Butler‡, Colette Cywes-Bentley§, and James M. Musser*¶

*Center for Molecular and Translational Human Infectious Diseases Research, Methodist Hospital Research Institute, Houston, TX 77030; ‡Southwest Foundation for Biomedical Research, San Antonio, TX 78227; and §Channing Laboratory, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115

Communicated by Richard M. Krause, National Institutes of Health, Bethesda, MD, September 1, 2006 (received for review July 20, 2006) The molecular basis of pathogen clone emergence is relatively frequency and severity of serotype M3 invasive infections (3). poorly understood. Acquisition of a bacteriophage encoding a Humans with GAS infections seroconvert to SlaA, indicating previously unknown secreted phospholipase A2 (designated SlaA) that this protein is made during infection (3). In addition, SlaA has been implicated in the rapid emergence in the mid-1980s of a has enzymatic activity against several phospholipid head groups new hypervirulent clone of serotype M3 group A Streptococcus. and acyl chains located at the sn-2 position (14). For example, Although several lines of circumstantial evidence suggest that SlaA SlaA cleaves and releases arachidonic acid, a potent mediator of is a virulence factor, this issue has not been addressed experimen- the inflammatory cascade. tally. We found that an isogenic ⌬slaA mutant strain was signifi- With the goal of directly testing the hypothesis that SlaA cantly impaired in ability to adhere to and kill human epithelial cells contributes to pathogenesis, we made a ⌬slaA isogenic mutant compared with the wild-type parental strain. The mutant strain strain from a wild-type serotype M3 strain and studied its role was less virulent for mice than the wild-type strain, and immuni- in GAS host–cell interaction and contribution to virulence in zation with purified SlaA significantly protected mice from invasive mouse and monkey models of infection. disease. Importantly, the mutant strain was significantly attenuated for colonization in a monkey model of pharyngitis. We Results conclude that transductional acquisition of the ability of a GAS Exogenously Presented SlaA Is Not Cytotoxic to Cultured Human strain to produce SlaA enhanced the spread and virulence of the Epithelial Cells. SlaA has PLA2 activity in vitro against several serotype M3 precursor strain. Hence, these studies identified a physiologically relevant substrates present in host cell mem- crucial molecular event underlying the evolution, rapid emergence, branes (14). Degradation of phospholipids by phospholipases and widespread dissemination of unusually severe human infec- can damage host membranes and decrease cell viability. To tions caused by a distinct bacterial clone.

determine whether exogenous SlaA caused host-cell cytotoxic- MICROBIOLOGY ity, purified recombinant SlaA (rSlaA) was incubated with bacteria ͉ Group A Streptococcus ͉ Streptococcus pyogenes immortalized pharyngeal epithelial (D562) cells, and lactate dehydrogenase (LDH) released into the culture medium was lthough of tremendous concern to society and public health measured as an indicator of cell damage. No significant increase Aauthorities, the molecular events, epidemiological pro- in LDH activity was detected (data not shown). Similarly, rSlaA cesses, and host factors that contribute to rapid emergence of did not have a detrimental effect on host-cell morphology or new pathogenic bacterial clones are poorly understood (1, 2). membrane integrity, as assessed by transmission electron mi- Information about these processes is needed to develop a croscopy (Fig. 7, which is published as supporting information on predictive model of bacterial epidemics and new drugs, vaccines, the PNAS web site). These results indicated that exogenous and diagnostics. To better understand clone emergence and rSlaA alone does not produce a substantive cytotoxic effect on changes in disease frequency and severity, we have used genome- human epithelial cells grown in vitro. wide analysis methods to study group A Streptococcus (GAS), a model human pathogen (3–6). GAS causes infections ranging in SlaA Increases GAS Attachment to and Killing of Human Epithelial severity from relatively mild pharyngitis and skin infections to Cells. To determine whether SlaA contributes to pathogenesis, life-ending invasive diseases such as septicemia, necrotizing we made a ⌬slaA isogenic mutant strain from a wild-type fasciitis, and streptococcal toxic shock syndrome (7). serotype M3 strain (Figs. 8 and 9, which are published as Serotype M3 strains have been of particular interest, because supporting information on the PNAS web site). We tested the comprehensive population-based studies have shown that these hypothesis that SlaA production influenced GAS–host cell organisms cause a disproportionate number of severe invasive interaction by using D562 cells and normal human trancheo- disease infections, such as necrotizing fasciitis and death (8–11). bronchial epithelial (NHTBE) cells. In both cell types, signifi- Based on genome sequencing and molecular population genetic analysis of strains recovered over Ͼ60 years, we discovered that Author contributions: I.S. and J.M.M. designed research; I.S., M.J.N., P.S., S.D.B., and C.C.-B. acquisition of a bacteriophage encoding a new secreted phos- performed research; I.S., M.J.N., C.C.-B., and J.M.M. analyzed data; and I.S. and J.M.M. pholipase A2 (PLA2) named SlaA created a new clone that now wrote the paper. is responsible for the vast majority of human infections caused The authors declare no conﬂict of interest. by serotype M3 strains in many countries (3, 4, 12). SlaA is Abbreviations: GAS, Group A Streptococcus; PLA2, phospholipase A2; SlaA, streptococcal secreted extracellularly and is related to a potent toxin (textilo- phospholipase A; rSlaA, recombinant SlaA; NHTBE, normal human tracheobronchial epi- toxin) made by the Australian common brown snake, Pseudonaja thelial; SLO, streptolysin O; moi, multiplicity of infection. textilis (3, 13, 14). †Present address: Department of Pharmacology, University of North Carolina, Several lines of evidence suggest that SlaA is a GAS virulence Chapel Hill, NC 27599. factor. The slaA gene was not present in serotype M3 strains until ¶To whom correspondence should be addressed. E-mail: [email protected]. the mid-1980s, a time frame that correlated with the increase in © 2006 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607669103 PNAS ͉ October 24, 2006 ͉ vol. 103 ͉ no. 43 ͉ 16009–16014 Downloaded by guest on October 2, 2021 A A >100 B P=0.009 100 2 1 80 WT 0 IgG 60 -2 control

40 -4 B 20 -6 Attached bacteria per cell

10:1 30:1 300:1 Fold change in attachment -8 Anti-SlaA Ratio of GAS/D562 cells antibody

3.0 P=0.034 P=0.045 C D 12 70 C P=0.02 2.5 10 60 8 50 40 2.0 6 n.s. 30 1.5 4 20 Fold change in attachment

% of dead cells 2 10 1.0 0 0 WT D562 cells NHTBE cells P=0.0005 +rSlaA WT D 35 WT 30 Fig. 2. Characterization of SlaA-dependent GAS–human cell interactions. (A) Adherence of the wild-type strain is dose-dependent, whereas adherence of the 25 P=0.004 ⌬slaA mutant strain is not. Experiments were performed with D562 cells as described but at the indicated mois. (B) Depletion of SlaA decreases GAS adher- 20 ence to D562 cells. SlaA was titrated with 10 ␮g of anti-SlaA antibody during 15 infection with wild-type MGAS315. Affinity-purified preimmune rabbit IgG at the same concentration was used as a control. (C) Addition of purified rSlaA to the 10 infection assay increased adherence of the ⌬slaA mutant strain. rSlaA (50 ␮g) was 5 added simultaneously with the wild-type and ⌬slaA mutant strains. n.s., not Attached bacteria per cell significant. (D) Production of SlaA significantly enhanced GAS killing of human 0 epithelial cells. D562 or NHTBE epithelial cells were cocultured with wild-type or D562 cells NHTBE cells the ⌬slaA isogenic mutant strain (moi ϭ 100:1, 3 h) and stained with trypan blue. The number of stained cells in 50 randomly chosen fields was counted by light Fig. 1. Production of SlaA significantly enhances adherence of GAS to microscopy, and the number of positive cells was expressed as percent of total human epithelial cells. D562 and NHTBE cells were infected with wild-type or cells in the field. P values were determined by t test of data for mutant vs. the ⌬slaA mutant strain (moi ϭ 100:1, 3 h), washed with PBS, fixed, and stained wild-type. NHTBE are primary cells and are much more susceptible to killing by with crystal violet. Photomicrographs for NHTBE cells are not shown, but the GAS than the immortalized D562 cells. results mirrored the D562 data. (A) Uninfected D562 cells. (B) D562 cells infected with wild-type strain MGAS315. (C) D562 cells infected with ⌬slaA isogenic mutant strain. (Magnification, ϫ40.) (D) Quantitative differences in host-cell adherence between the wild-type and ⌬slaA mutant strains by using type GAS, indicating that SlaA gained access to the host cell D562 cells (Left) and NHTBE cells (Right). The number of cell-associated cytoplasm (Fig. 3A). These results were confirmed with confocal bacteria was determined by counting GAS attached to 50 randomly selected microscopy (Fig. 3 B and C). Taken together, the data suggest human cells in five different microscope fields. The results are expressed as the that SlaA plays an important role in facilitating GAS adherence mean number of GAS per host cell. An enlarged version of Fig. 1 is presented to cultured epithelial cells, and that entry of SlaA into host cells as Fig. 10, which is published as supporting information on the PNAS web site. is required for cytotoxicity. To investigate the mode of SlaA entry into host cells, we tested the hypothesis that transmembrane pores generated by GAS cantly fewer ⌬slaA GAS were associated with the host cells streptolysin O (SLO) were involved. Pore formation by the GAS compared with the wild-type strain (Fig. 1). Importantly, de- cytolytic toxin SLO has been postulated to be equivalent to type creased attachment could not be overcome by adding more III secretion in Gram-negative bacteria (17). NHTBE cells were mutant bacteria (Fig. 2A). The number of wild-type GAS incubated with serotype M3 strain 950771SmsloϪ, which pro- attached to epithelial cells increased proportionally with an slo ͞ ⌬ duces SlaA but not SLO, because of deletion of the structural increased cfu cell ratio, an effect not observed with the slaA gene (18). SlaA was present in the cytosolic fractions prepared mutant strain. Depletion of SlaA by addition of anti-SlaA from ⌬slo-infected host cells (Fig. 3D, lane 2), suggesting that antibody significantly decreased GAS adherence to host cells SlaA does not require SLO to gain access to the host cell cytosol. (Fig. 2B), and addition of exogenous rSlaA increased the In contrast, cytochalasin D blocked transport of SlaA into ⌬ Ͼ number of slaA mutant GAS attached to host cells by 2-fold human epithelial cells (Fig. 3D, lane 1). These results suggest that (Fig. 2C). Several reports indicate that GAS infection of epi- SlaA enters host cells by an active transport process requiring thelial cells results in apoptosis (15, 16). Thus, we hypothesized intact cytoskeleton function. that the ⌬slaA mutant strain would kill fewer host cells as a consequence of decreased adherence. We found that the ⌬slaA SlaA Is a Virulence Factor. The observation that serotype M3 GAS mutant killed significantly fewer host epithelial cells compared strains expressing SlaA are overrepresented among invasive with the wild-type strain (Fig. 2D). disease isolates (3, 4, 14, 19) suggests that SlaA contributes to To determine whether SlaA or SlaA-producing bacteria en- GAS pathogenesis. In addition, patients with pharyngitis or tered the host cell, NHTBE cells were incubated with wild-type invasive infections seroconvert to SlaA, indicating that this or ⌬slaA mutant GAS, and cell lysates were analyzed by Western enzyme is secreted during infection (3). To determine whether immunoblotting. Immunoreactive SlaA was found in the cell SlaA plays a role in GAS invasive infection, we compared the membrane and cytosolic fractions of cells incubated with wild- ability of the wild-type and ⌬slaA mutant strains to cause mouse

16010 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607669103 Sitkiewicz et al. Downloaded by guest on October 2, 2021 A B C

x-z y-z

D 250 200 150 100 50 0 53035404510 15 20 25 Fluorescence intensity Cell width (mm)

Fig. 3. NHTBE cells were infected with wild-type GAS (lanes 1, 3, and 5) or the ⌬slaA mutant strain (lanes 2, 4, and 6), fractionated, and analyzed by Western immunoblot with anti-SlaA antisera. Lanes 1 and 2, cell culture medium; lanes 3 and 4, NHTBE membrane fraction; lanes 5 and 6, NHTBE cytosolic fraction.(B) Confocal microscopy images of NHTBE cells 6 h after infection with wild-type GAS. Cell membranes were labeled with anti-CD44 antibody (red), and SlaA was visualized with Alexa 488-conjugated secondary antibody (green, green arrows). White arrowheads indicate the cell membrane. x-z and y-z crosssections through the cell layer (black arrowheads) are shown in Upper and Right, respectively. (C) SlaA enters epithelial cells. The dashed white line corresponds to the crosssection position. The cells were imaged in 0.5-␮m sections from the apical to the basal surface (z-stack). A pixel intensity projection in the x-y orientation indicates the red peaks of the cell-surface CD44 staining (red arrowheads) and green SlaA intracellular peaks (green arrowheads). The red and green baselines represent background staining. (D) Intracellular transport of SlaA is streptolysin O-independent and cytochalasin D-dependent. Western immunoblot analysis of SlaA in cytoplasmic fractions of NHTBE cells after infection with a ⌬slo mutant strain (lane 2) or after infection with the same strain in the presence of cytochalasin D (lane 1).

near mortality after i.p. inoculation. Significantly fewer mice after s.c. inoculation compared with the wild-type strain (Fig. 4 infected with the ⌬slaA isogenic mutant strain reached near B and C). Western immunoblot analysis of proteins extracted mortality compared with animals infected with the wild-type from skin lesions confirmed that SlaA was made by the wild-type parental strain (Fig. 4A). One characteristic of serotype M3 strain at the site of infection (Fig. 4D). human infections is unusually severe tissue destruction, as exemplified by necrotizing fasciitis (8, 10, 11). Important to note, Protection of Mice by Immunization with Recombinant SlaA. Inas- the ⌬slaA mutant strain also caused significantly less morbidity much as SlaA is produced during infection of humans and mice MICROBIOLOGY

100 100 A WT B 75 75 P=0.001 n=16 mice per group WT 50 50

25 25 P=0.013 Percent survival

Percent survival Percent n=16 mice per group 0 0 0255075100125 0 5 10 15 20 25 30 Hours after infection Days after infection

1500 12 34 C D kDa 54

3 1250 WT n=30 mice ∆slaA n=31 mice 37 1000 29 P=0.027 750 20 500

250

Abscess volume volume (mm ) Abscess 7 0 25 9 16 Days after infection

Fig. 4. SlaA contributes to GAS virulence in both septicemia and skin-lesion mouse models of infection. CD-1 Swiss male mice were inoculated i.p. with 2.5 ϫ 107 cfu of wild-type MGAS315 or the ⌬slaA isogenic mutant strain. Immunocompetent hairless mice (strain Clr:SKH1-hrBR) were s.c.-inoculated with 1 ϫ 107 cfu. Kaplan–Meier survival curves were plotted to compare the difference in near mortality after i.p. (A) and s.c. (B) inoculation. (C) ⌬slaA mutant strain induced significantly smaller lesions in the s.c. model of infection. Average lesion volumes were compared for the wild-type and ⌬slaA mutant strains at various days postinfection. (D) Western immunoblot analysis showing production of SlaA at the lesion site. Mice were inoculated with wild-type or ⌬slaA mutant strains, killed at 48 h postinfection, and tissue at the lesion site was excised. Proteins were extracted from the tissue and analyzed by Western immunoblot by using SlaA-specific polyclonal rabbit antibody. Lane 1, purified recombinant SlaA; lane 2, extract from wild-type-infected mice; lane 3, extract from ⌬slaA-infected mice to which purified recombinant SlaA was added before extraction (extraction control); lane 4, extract from ⌬slaA-infected mice.

Sitkiewicz et al. PNAS ͉ October 24, 2006 ͉ vol. 103 ͉ no. 43 ͉ 16011 Downloaded by guest on October 2, 2021 Fig. 5. Immunization of mice with SlaA abrogates GAS pathogenesis. (A) Immunocompetent hairless mice (strain Clr:SKH1-hrBR) were immunized with SlaA, boosted, and challenged with wild-type strain MGAS315. Lesion volumes were calculated, and averages were compared for unimmunized and SlaA-immunized animals at the indicated times postinfection. The difference in abscess volumes between immunized and sham-immunized control animals was statistically significant, as assessed by mixed-model repeated measures. (B) Representative lesions induced by wild-type GAS after s.c. inoculation of immunized mice. (Left) Sham-immunized mice. (Right) SlaA-immunized mice. (C) Increased ␣-SlaA antibody titers correlated with smaller lesion size upon GAS challenge. Serum was obtained from immunized mice 2 days before challenge with strain MGAS315. Antibody levels were determined by ELISA with purified rSlaA. Statistical significance was tested by the Pearson correlation test (Pearson r ϭϪ0.585; 95% confidence interval, Ϫ0.859 to Ϫ0.050).

(ref. 3 and Fig. 4D) and contributes to virulence, it is possible monas aeruginosa (29–32), which is delivered directly to the host that immunization with rSlaA would protect against GAS dis- cell cytosol by the type III secretion machinery. As observed with ease. Mice immunized with SlaA had significantly smaller ab- SlaA, ExoU lacks cytotoxic effect when added exogenously to scesses compared with sham-immunized control animals (Fig. 5 host cells grown in vitro; however, intracellular ExoU has a rapid A and B). In addition, mice with high levels of anti-SlaA antibody and profound cytotoxic effect on cultured cells (29). The idea had significantly smaller skin lesions compared with animals that SlaA acts from within host cells was further supported by with low anti-SlaA antibody levels (Fig. 5C). These results data showing that expression of SlaA in yeast results in decreased further support the idea that SlaA contributes to the enhanced host-cell viability (Fig. 11, which is published as supporting virulence of serotype M3 strains containing the slaA gene. information on the PNAS web site). In addition, exhaustive attempts to clone the gene for genetic complementation of the SlaA Significantly Enhances Upper Respiratory Tract Infection. Large epidemiological studies have shown that genotypes of GAS strains that are abundant causes of tonsillitis (‘‘strep throat’’) A WT also are the dominant types causing invasive episodes in the same 6 10 ∆ geographic area (20, 21). Recently, the cynomolgus macaque has slaA become the gold standard for experimental GAS pharyngitis 10 5 studies (22, 23). Thus, we tested the hypothesis that SlaA contributed to colonization and infection in this model. The 10 4 mutant strain was strikingly attenuated for infection and persis- CFU/swab tence (Fig. 6A). Furthermore, animals infected with the wild- 10 3

type strain seroconverted to SlaA (Fig. 6B), thus confirming that bacterial titer Average SlaA was expressed in vivo in the upper respiratory tract. 10 2 2 4 7 11 15 18 21 28 Discussion Day post-inoculation Although of great interest to many fields of biomedical research, the molecular genetic events contributing to the creation and 4 very rapid emergence of new pathogenic bacterial clones are B poorly understood (1, 2). GAS is an ideal model organism to WT study these processes, because population-based strain collec- 3 ∆ tions are available that are linked to detailed clinical informa- slaA

tion. In addition, the organism is known to exhibit precipitous 450

changes in disease frequency and severity, and the genome OD 2 sequences of 12 strains are available (3, 6, 12, 19, 24–28). The present study was motivated by several lines of circumstantial 1 evidence that together implicated acquisition of a bacteriophage encoding SlaA as a key event underlying the recent and rapid emergence of a new unusually virulent serotype M3 clone (3, 4, 0 12). Our studies unambiguously demonstrate that SlaA is an -7 71521 28 PC important GAS virulence factor, thereby providing considerable Day support to the hypothesis that it contributed to clone emergence and the observed increased severity of serotype M3 infections. Fig. 6. SlaA is required for GAS infection of cynomolgus macaques. Two groups of four monkeys were infected with wild-type (WT) strain MGAS315 or Although we did not investigate the precise host substrate(s) the ⌬slaA isogenic mutant strain and monitored. (A) The tonsils of monkeys for SlaA, the epithelial cell culture data suggest that the enzyme were swabbed at the indicated time points, and recovered bacteria were exerts its cytotoxic effects intracellularly. In this regard, SlaA has plated to determine cfus. (B) Monkey sera from the indicated time points were parallels with ExoU, a PLA2 virulence factor made by Pseudo- analyzed by ELISA for anti-SlaA antibody. PC, positive control.

16012 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0607669103 Sitkiewicz et al. Downloaded by guest on October 2, 2021 mutant strain failed, consistent with the idea that SlaA is Jose, CA). NHTBE cells (Clonetics, San Diego, CA) were grown cytotoxic when expressed within cells (see Supporting Text, which on collagen-coated glass cover slips in 24-well plates (Corning, is published as supporting information on the PNAS web site). Corning, NY) to 80–90% confluency in BEBM medium with It is a formal possibility that SlaA itself is not directly cytotoxic supplements as recommended by the manufacturer (Clonetics) to human cells; rather, it may be that increased host-cell death at 37°C with 5% CO2. occurs secondary to the enhanced adherence of GAS mediated by SlaA. However, the yeast expression data favor the idea that Construction of the ⌬slaA Isogenic Mutant Strain. Details are pro- SlaA is directly cytotoxic, although both processes may be vided in Supporting Text. We exhaustively attempted to geneti- operative. cally complement the slaA deletion strain by introducing a We also found that SlaA was secreted by GAS at the site of wild-type copy of slaA into the mutant strain. Details are soft-tissue infection. Inactivation of the slaA gene resulted in provided in Supporting Text. significantly decreased virulence of the mutant strain in two mouse models of GAS invasive infection, and immunization with Purification of SlaA. rSlaA was purified to homogeneity from E. SlaA significantly decreased the amount of tissue pathology coli and tested for PLA2 activity (ref. 3 and Supporting Text). observed in mice after s.c. inoculation. These findings lead us to believe that SlaA enhances the pathogenic processes occurring In Vitro Infection, Attachment, and Cell Death Assays. NHTBE cells during deep-tissue infection in the human, either directly or were grown as previously described. Growth media were re- indirectly. placed the morning of infection. D562 epithelial cells were grown Thus, our data suggest a model in which SlaA acts in a in MEM medium with serum for 48 h after seeding, and the multifaceted manner by enhancing host colonization and tissue media were replaced with serum-free MEM on the day of destruction, which together would increase the number and infection. Overnight GAS cultures were diluted into THY severity of invasive infections caused by serotype M3 strains. medium and grown to an OD600 Ϸ0.3. The bacteria were SlaA increased the adherence of GAS to human epithelial cells, pelleted, washed with PBS, and suspended in PBS to an OD600 the first stage of host–pathogen cellular interaction. The ability of Ϸ2.0. A 50-␮l aliquot [resulting in an multiplicity of infection of SlaA to enhance GAS adherence to epithelial cells would (moi) of 100:1] was added to each well of human cells and expand the population size and the possibility of transmission of incubated for 3 h. Nonadherent GAS were removed by washing these strains, thereby increasing the probability that SlaA- with PBS. For cytotoxicity assays, host cells were stained for 10 producing organisms would encounter a host susceptible to, or min with trypan blue (1:10 dilution in PBS), fixed in 2% at risk for, invasive GAS infection. In this regard, we note that paraformaldehyde (1 h), and the mean percentage of stained SlaA production is greatly increased when GAS interacts with cells was determined. To determine the number of cell- human saliva or respiratory tract epithelial cells (13, 14, 33). associated bacteria, fixed cells were stained overnight with Consistent with these findings, patients with GAS tonsillitis crystal violet, and the number of GAS attached to 50 randomly seroconvert to SlaA (3), which means that this enzyme is made selected human cells in five different microscopic fields was during human upper respiratory tract infection. Together, these determined. Lactate dehydrogenase release assays were per- observations strongly suggest that SlaA is secreted very early in formed per the manufacturer’s instructions (CytoTox-ONE;

host–pathogen interaction, an optimal time to establish infection Promega, Madison, WI). MICROBIOLOGY and drive clonal expansion. Antibody-Inhibition Assay. The attachment assay was performed as Concluding Comment. To summarize, many social, political, and described above, except that 10 ␮g of affinity-purified rabbit economic factors contribute to the emergence and reemergence anti-SlaA antibody was added to the D562 cells immediately of infectious diseases (2). However, with the exception of the before addition of bacteria. Affinity-purified preimmune rabbit acquisition of genes conferring antimicrobial agent resistance, IgG at the same concentration was used as a control. relatively little information is available about the molecular processes contributing to the very rapid emergence of distinct Host-Cell Localization of SlaA. For immunological localization of pathogenic bacterial clones. The sum of the evidence points to SlaA, NHTBE cells were infected for 2.5 h (moi ϭ 100:1). Anti- a model in which very recent acquisition of the gene encoding biotics (100 ␮g͞ml clindamycin and 10 ␮g͞ml penicillin) were then SlaA created a new clone of serotype M3 GAS with significantly added for 30 min. Cytochalasin D (70 ␮g͞ml) was added to selected enhanced epithelial cell colonization capacity and unusually high samples during the infection. Epithelial cells were detached with virulence traits. trypsin-EDTA, collected by centrifugation, washed, and lysed by incubation with 1 mg͞ml digitonin. Cell lysates were centrifuged at Materials and Methods 20,800 ϫ g for 5 min to yield a cytosolic and membrane fraction. The Bacterial Strains, Culture Conditions, and DNA Manipulation. The samples were analyzed by Western immunoblot with rabbit anti- bacterial strains and plasmids used in this study are listed in SlaA antibody (1:10,000 dilution) followed by alkaline phosphatase- Table 1, which is published as supporting information on the conjugated goat anti-rabbit antibody (1:5,000; Santa Cruz Biotech- PNAS web site. GAS strains were grown in Todd–Hewitt broth nology, Santa Cruz, CA). (Difco, Sparks, MD) supplemented with 0.2% yeast extract For cell localization of SlaA using confocal microscopy, (THY medium) at 37°C in 5% CO2͞20% O2 atmosphere. THY NHTBE cells were infected for 6 h (moi ϭ 100:1). Infected medium or tryptose agar with 5% sheep blood (Becton Dick- NHTBE cells were then washed with PBS and fixed with 4% inson, Franklin Lakes, NJ) was used as solid medium. Cloning paraformaldehyde in PBS (pH 7.4), and host cell membranes experiments were performed with Escherichia coli Nova Blue were labeled with 5 ␮g͞ml Alexa 568-conjugated anti-CD44 (Novagen, San Diego, CA). Ampicillin (100 ␮g͞ml) and spec- antibody (IM7.8.1; BD Biosciences). To visualize intracellular tinomycin (150 ␮g͞ml) were added when applicable. SlaA, CD44-labeled cells were washed with PBS, permeabilized with 0.1% Triton X-100 in PBS, and incubated with primary Human Epithelial Cell Culture. Immortalized Detroit 562 (D562) rabbit anti-SlaA antibodies followed by secondary staining with pharyngeal epithelial cells were purchased from the American Alexa 488-conjugated donkey anti-rabbit IgG (Molecular Type Culture Collection (Manassas, VA; CCL-138). The cells Probes, Eugene, OR). Confocal microscopy images were col- were grown to 80–90% confluency in MEM (Invitrogen, Carls- lected by using a Zeiss (Oberkochen, Germany) LSM 510 Pascal bad, CA) supplemented with 10% FBS (BD Biosciences, San confocal microscope equipped with an Argon͞Krypton and two

Sitkiewicz et al. PNAS ͉ October 24, 2006 ͉ vol. 103 ͉ no. 43 ͉ 16013 Downloaded by guest on October 2, 2021 HeNe lasers (Zeiss) and images processed by using Zeiss anti-SlaA antibody (3). ELISA on mouse sera (1:2,000 dilution) software. from SlaA-vaccinated animals was done by standard procedures with purified SlaA as antigen. ELISA on monkey sera (1:500 Mouse Infection Experiments. GAS strains used for infection dilution) was done by standard procedures with purified SlaA as studies were grown in THY medium to mid-exponential phase antigen. (OD600 Ϸ0.5), harvested, washed twice with PBS, and used to infect either immunocompetent hairless mice (strain Crl:SKH1- Non-Human Primate Infection. A non-human primate model of hrBR; s.c. inoculation, administered dose 1.0 ϫ 107 cfu) or CD-1 GAS pharyngitis was used (22, 23). Two groups of four anes- Swiss mice (i.p. inoculation, administered dose 2.5 ϫ 107 cfu). Abscess length (L) and width (W) values were used to calculate thetized cynomolgus macaques were inoculated with a 1-ml ϫ 7͞ ⌬ abscess volume (V ϭ 4͞3␲ (L͞2)2 ϫ [W͞2]) and area (A ϭ suspension (8 10 ml) of wild-type and slaA GAS strains into ␲[L͞2] ϫ [W͞2]) by using equations for a spherical ellipsoid (34). the nares of each animal. Throat swabs and peripheral blood samples were taken on days Ϫ7, 7, 15, 21, and 28. Throat swabs Immunization of Mice with rSlaA. Crl:SKH1-hrBR mice were vac- only were taken on days 0, 2, 4, 11, and 18. cinated with purified rSlaA (50 ␮g) and TiterMax adjuvant (TiterMax USA, Norcross, GA). Control mice were injected with We thank R. Ireland, I. Abdi, D. Dorward, R. Larson, J. Rosenblatt, K. PBS and TiterMax. At 4 weeks postimmunization, mice were Ponce, A. Raiford, and M. Strauss for technical assistance; T. Quitugua boosted with 25 ␮g of rSlaA. Two weeks later, they were for generous hospitality; K. Stockbauer for editorial help; E. A. Graviss challenged by s.c. injection as described above. for help with statistical analysis; and R. M. Krause, D. Morens, and F. R. DeLeo for critical comments to improve the manuscript. This work was Serologic Analysis of SlaA. In vitro production of SlaA was assessed supported in part by Southwest National Primate Research Center Pilot by Western immunoblot analysis with specific rabbit polyclonal Study Grant 05-3500-77.

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