The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Voyich, J. M.
Right arrow Articles by DeLeo, F. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Voyich, J. M.
Right arrow Articles by DeLeo, F. R.
The Journal of Immunology, 2004, 173: 1194-1201.
Copyright © 2004 by The American Association of Immunologists

Engagement of the Pathogen Survival Response Used by Group A Streptococcus to Avert Destruction by Innate Host Defense

Jovanka M. Voyich, Kevin R. Braughton, Daniel E. Sturdevant, Cuong Vuong, Scott D. Kobayashi, Stephen F. Porcella, Michael Otto, James M. Musser and Frank R. DeLeo1

Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neutrophils are a critical component of human innate host defense and efficiently kill the vast majority of invading microorganisms. However, bacterial pathogens such as group A Streptococcus (GAS) successfully avert destruction by neutrophils to cause human infections. Relatively little is known about how pathogens detect components of the innate immune system to respond and survive within the host. In this study, we show that inactivation of a two-component gene regulatory system designated Ihk-Irr significantly attenuates streptococcal virulence in mouse models of soft tissue infection and bacteremia. Microarray analysis of wild-type and irr-negative mutant (irr mutant) GAS strains revealed that Ihk-Irr influenced expression of 20% of all transcripts in the pathogen genome. Notably, at least 11 genes involved in cell wall synthesis, turnover, and/or modification were down-regulated in the irr mutant strain. Compared with the wild-type strain, significantly more of the irr mutant strain was killed by human neutrophil components that destroy bacteria by targeting the cell envelope (cell wall and/or membrane). Unexpectedly, expression of ihk and irr was dramatically increased in the wild-type strain exposed to these same neutrophil products under conditions that favored cell envelope damage. We report a GAS mechanism for detection of innate host defense that initiates the pathogen survival response, in which cell wall synthesis is critical. Importantly, our studies identify specific genes in the pathogen survival response as potential targets to control human infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Neutrophils are essential effector cells of the human innate immune system and provide the primary defense against invading microorganisms. Phagocytosis of bacteria elicits production of reactive oxygen species (ROS)2 and release of antibacterial granule components into pathogen-containing phagocytic vacuoles. Although neutrophil ROS and granule components are highly effective at killing most bacteria, some human pathogens evade neutrophil phagocytosis to survive or, alternatively, avert destruction after phagocytosis (1, 2, 3).

Streptococcus pyogenes (group A Streptococcus (GAS)) circumvents killing by neutrophils to cause human infections. The ability of GAS to evade neutrophil-mediated killing includes inhibition of phagocytosis, new mechanisms for which have been discovered recently (4, 5, 6). In addition, current evidence suggests the pathogen is resistant to the effects of neutrophil antibacterial components following phagocytosis (1, 7, 8, 9, 10). We previously used GAS microarrays to identify a complex genetic program used by the pathogen to escape destruction by neutrophils (7). The GAS genome-wide protective response revealed by those studies included up-regulation of ihk and irr, genes encoding a two-component gene regulatory system named Ihk-Irr (7). Notably, GAS lacking irr are rapidly destroyed after phagocytosis, but the molecular basis of this phenotype is not known. Inasmuch as the ability of pathogenic microbes to withstand neutrophil killing is central to their ability to cause disease, it is crucial to understand host factors that trigger the pathogen survival response. We report a mechanism used by GAS to detect and evade human innate immunity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Todd-Hewitt broth containing 0.2% yeast extract (THY) was obtained from BD Biosciences (Sparks, MD). Sterile water and 0.9% sodium chloride (both Irrigation USP; Baxter Healthcare, Deerfield, IL) were purchased from Baxter Healthcare. Dextran T-500 and Ficoll-Paque PLUS were obtained from Amersham Biosciences (Piscataway, NJ). RPMI 1640 medium was purchased from Invitrogen Life Technologies (Carlsbad, CA). Unless specified, all other reagents were from Sigma-Aldrich (St. Louis, MO).

Bacterial strains and culture

For in vitro microarray analyses, serotype M6 GAS strain JRS4 (wild-type) and isogenic irr-negative mutant strain JRS500 (irr mutant) (11) were grown in THY to early exponential phase (OD600 = 0.35), or late exponential phase (OD600 = 0.75), and harvested immediately for RNA isolation. For all other assays, GAS strains were grown to early exponential phase, washed in Dulbecco’s PBS (DPBS), and resuspended in RPMI 1640 medium containing 10 mM HEPES (RPMI/H) at 109 per milliliter. GAS strains representative of those causing invasive (serotype M1, SF370) (12) or noninvasive (serotype M18, MGAS8232) (13) disease in humans were cultured as described above.

Isolation of human neutrophils

Neutrophils were isolated from heparinized venous blood of healthy individuals with dextran sedimentation followed by Ficoll-Paque PLUS (Amersham Biosciences) gradient separation as described previously (7, 14). All studies with human blood were performed in accordance with a protocol approved by the Institutional Review Board for Human Subjects, National Institute of Allergy and Infectious Diseases.

Mice

Crl:SKH1-hrBR female (outbred immunocompetent, hairless) and CD1 Swiss mice were purchased from Charles River Laboratories (Willmington, MA). Animals were between 5 and 6 wk of age, housed in microisolator cages, and received food and water ad libitum. The studies conformed to guidelines set forth by the National Institutes of Health and were reviewed and approved by an Animal Use Committee at Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases.

Mouse infection models

Wild-type and irr mutant GAS strains were grown to early exponential phase, washed twice with sterile DPBS, and resuspended to 108 GAS/200 µl. Mice were anesthetized with isoflurane and inoculated by s.c. or i.p. injection with 108 wild-type or irr mutant GAS strains, or with sterile DPBS. There were eight mice in each treatment group for the soft tissue infection model (s.c. inoculation in the left shoulder). Size of each skin abscess was measured after initial inoculation with a sliding caliper and monitored daily for 14 days. The bacteremia experiments were performed two separate times. There were 16 mice inoculated by i.p. injection with the wild-type strain, 15 mice inoculated with the irr mutant strain, and 15 mice inoculated with sterile DPBS. Animals were bled via the saphenous vein before i.p. inoculation and at 5- and 24-h postinoculation. Blood samples were cultured overnight on blood agar plates, and animals from which GAS was cultured were considered positive for bacteremia.

GAS microarray experiments

Microarray experiments were performed as previously described with few modifications (7). Wild-type and irr mutant GAS strains were cultured to early and late exponential phases of growth as described above. We performed microarray experiments with early and late exponential-phase GAS cultures (cultures were not combined, and each culture was assayed independently), because it is unclear which phase of in vitro growth best represents that of in vivo bacterial infection. A total of 2.5 x 109 bacteria was lysed with 700 µl of RLT buffer (Qiagen, Valencia, CA) and the lysate homogenized with an FP120 FastPrep system (Qbiogene, Carlsbad, CA). Total RNA was isolated with RNeasy kits (Qiagen). First-strand cDNA synthesis (incorporation of biotin and fluorescein probes) and tyramide signal amplification (TSA) was performed for RNA samples using Micromax TSA Labeling and Detection kit (PerkinElmer Life Sciences, Shelton, CT) with some modification of the manufacturer’s protocol. Random primers at 0.5 µg/ml (Promega, Madison, WI) were added to the cDNA reaction mixture. The RNA/cDNA hybridization mixtures were denatured by NaOH hydrolysis (0.3 M final concentration) at 65°C for 15 min. The cDNA was purified by using QIAquick PCR Purification kit (Qiagen) with 75% ethanol substituted for the wash buffer and nuclease-free water substituted for the elution buffer. Biotin- and fluorescein-labeled cDNA samples were combined (50 µl total), diluted 50% with SlideHyb 3 (Ambion, Austin, TX), denatured at 95°C for 2 min, and incubated with a DNA microarray containing 1705 (of 1752) M1 GAS open reading frames (ORFs) based on strain SF370 (12), and unique M18 and M3 ORFs (13, 15). PCR products derived from each ORF were printed onto CMT-GAPS Corning (Acton, MA) glass slides with a Chipwriter robotic arrayer (Bio-Rad, Hercules, CA). Hybridization of cDNA samples to microarray slides was conducted overnight in a 45°C water bath. Slides were washed sequentially with 0.5x SSC containing 0.1% SDS (twice), 0.06x SSC containing 0.01% SDS, and 0.06x SSC. Slide signal was amplified with TSA, and slides were scanned with a ScanArray 5000 instrument (PE Biosystems, San Diego, CA). Slides were normalized to equal fluorescence intensity against serial dilutions of MGAS8232 genomic DNA standards by adjusting laser power and/or photomultiplier gain. Spot location and array alignment were adjusted with QuantArray (PE Biosystems). All genes identified as differentially expressed passed a general filter based on signal-minus-background, spot area and signal-to-noise ratio, and were at least 1 SD above controls. To compare gene expression between the wild-type and irr mutant GAS strains, fold-changes for each gene were determined by the ratio of median fluorescence intensity of microarray spots derived from RNA samples of wild-type and irr mutant strains. Analysis for microarrays was done with GENESPRING software, version 4.2 (Silicon Genetics, Redwood City, CA). Microarray experiments that directly compared wild-type and mutant strains were performed in duplicate. Each microarray slide contained 4–12 spots per gene for a total of ≥8 spots for each gene. A link to the Gene Expression Omnibus (GEO) containing the complete set of microarray results compliant with Minimum Information about a Microarray Experiment guidelines can be found at www.niaid.nih.gov/dir/labs/lhbp/deleo.htm.

H2O2 and neutrophil granule killing assays

Serotype M6 wild-type and irr mutant GAS strains (108) were incubated in 96-well plates with varied concentrations of H2O2 for 60 min at 37°C as indicated. GAS were plated on THY agar, and colonies were enumerated the following day. The percentage of GAS killed was calculated using the following equation: (1 – (CFU+hydrogen peroxide/CFUcontrol)) x 100.

Subcellular fractionation of neutrophils was performed as previously described (16). Briefly, neutrophils were resuspended in relaxation buffer (100 mM KCl, 3 mM NaCl, 3.5 mM MgCl2, and 10 mM PIPES containing 1 mM ATP) and disrupted by nitrogen cavitation (400 psi for 20–30 min at 4°C). Neutrophil lysate was collected dropwise into 100 mM EGTA such that the final concentration was 1.25 mM. Lysates were centrifuged at 200 x g for 6 min to remove unbroken cells and nuclei. Neutrophil lysates were overlaid atop Percoll step gradients (16) and centrifuged at 48,300 x g for 15 min at 4°C. Fractions containing neutrophil primary ({alpha}) and secondary ({beta}) granules were collected as described (16), and Percoll was removed by ultracentrifugation at 100,000 x g for 90 min. To standardize bactericidal activity, aliquots of granule fractions were thawed once for use and then discarded. GAS strains were incubated with 106–107 neutrophil cell equivalents (as indicated) of each granule fraction for 60 min with 0.1% saponin and then plated on THY agar. Colonies were enumerated the following day and percentage of GAS killed was calculated using the following equation: (1 – (CFU+granules/CFUcontrol)) x 100.

Assays with antimicrobial peptides and proteins

GAS were cultured to early exponential growth phase and resuspended in RPMI/H to 105/ml. Peptides and proteins were reconstituted in buffer as recommended by the manufacturer: human {alpha}-defensin (Chemicon International, Temecula, CA), sterile water; neutrophil elastase (EMD Biosciences, San Diego, CA), 50 mM sodium acetate (pH 5.5) containing 200 mM NaCl; neutrophil cathepsin G (EMD Biosciences), 50 mM NaOAc, and 150 mM NaCl (pH 5.5); and LL-37 (Phoenix Pharmaceuticals, Belmont, CA), 10% acetonitrile with 0.1% trifluoroacetic acid. Peptide or enzyme dilution buffer (control) was added to each GAS strain, and samples were incubated at 37°C for 60 min. Samples were plated on THY and enumerated the following day with a ProtoCol SR colony counter system (Synbiosis, Frederick, MD). The percentage of GAS killed was calculated using the following equation: (1 – (CFU+peptide/CFUcontrol)) x 100.

TaqMan real-time RT-PCR analysis

GAS strains (2.5 x 108) were cultured to early exponential phase of growth and incubated with 1 mM H2O2 in THY medium for 30 or 60 min at 37°C. Alternatively, GAS were grown as described above, resuspended at 2.5 x 108/ml in RPMI/H containing 0.1% saponin, and incubated with 107 neutrophil equivalents of each granule fraction (as described above for granule killing assays) for 15, 30, and 60 min at 37°C with 5% CO2. At the indicated times, ihk and irr transcript levels were determined with TaqMan real-time RT-PCR (ABI 7700 thermocycler; Applied Biosystems, Foster City, CA) as described (7). Samples were assayed in triplicate from two to three biological replicates as indicated.

Statistics and curve fitting

Statistics were performed with a Student’s t test, logrank test, or ANOVA with a Tukey posttest for multiple comparisons using GraphPad Prism, version 4.0 for Windows (GraphPad Software, San Diego, CA), unless indicated otherwise. Curve fitting for the antibacterial peptide assays was performed with GraphPad Prism, version 4.0 for Windows, using Boltzmann sigmoidal nonlinear regression.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inactivation of irr significantly attenuates streptococcal virulence in mouse models of soft tissue infection and bacteremia

To determine whether Ihk-Irr is important for GAS infections in tissues, mice were inoculated with wild-type or irr mutant GAS strains by s.c. injection and monitored for several days (Fig. 1). There were notable differences in tissue infection caused by the two strains. First, abscesses formed more rapidly in mice infected with the wild-type strain (Fig. 1A, day 1). Second, the average abscess size was significantly larger in mice infected with the wild-type strain (p ≤ 0.017) (Fig. 1, A and B). Differences in development and size of abscesses between the two strains were not likely due to altered growth kinetics, because growth of the wild-type and irr mutant strains was essentially identical in vitro (Fig. 1C).



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 1. The Ihk-Irr two-component gene regulatory system is important for GAS pathogenesis. A, Cutaneous abscess size in individual mice infected by s.c. inoculation with wild-type (blue circles) or isogenic irr mutant (red triangles) strains of GAS. Black lines represent the average abscess size on each day. *, p < 0.017 vs irr mutant strain. **, p = 0.04 vs irr mutant strain. B, Representative abscesses 3 days after infection. C, In vitro growth of the wild-type (blue circles) and irr mutant (red triangles) GAS strains. D, Bacteremia in mice after i.p. inoculation of wild-type (blue circles) and irr mutant (red triangles) GAS strains. *, p = 0.03 vs wild-type strain.

 
We next compared the ability of the wild-type and irr mutant GAS strains to cause bacteremia (Fig. 1D). Mice were infected with each strain by i.p. inoculation, and bacteremia was assessed at 5- and 24-h postinfection (Fig. 1D). Five hours after inoculation, 12 of 16 mice infected with the wild-type strain and 11 of 15 mice infected with the irr mutant strain were bacteremic (Fig. 1D, left panel). However, 24 h after inoculation, only 1 animal infected with the irr mutant strain remained bacteremic, compared with 6 of those infected with the wild-type strain (p = 0.03 vs wild type) (Fig. 1D). Significantly accelerated clearance of GAS from the blood of animals inoculated with the irr mutant strain could reflect increased susceptibility of that strain to effectors of innate host defense such as neutrophils (7), or an attenuated ability to disseminate at the level of the wild-type strain. Nonetheless, the mouse infection data demonstrate conclusively that Ihk-Irr plays a key role in GAS pathogenesis.

Microarray analysis of genes influenced by ihk-irr

To gain insight into the molecular basis for differences in pathogenesis between the wild-type and irr mutant GAS strains, we used microarrays to compare global gene expression in each strain at early and late exponential phases of growth in vitro (Fig. 2A, Table I, and supplemental Table II, which contains the complete set of microarray data for these experiments). 3 Compared with the wild-type strain, there were significant changes in transcript lev els of 351 genes in the irr mutant strain (20% of the GAS genome) (Fig. 2A, Table I, and supplemental Table II). Notably, 218 of these genes were down-regulated in the irr mutant strain (Fig. 2A). For example, genes encoding transcription regulators such as codY (SPy1777), Spy0875, Spy1107, SPy1556, and a recently described regulator of CsrR/S known as rocA (SPy1605) (17) were down-regulated (Table I). A homolog of vicR (SPy0528) was up-regulated in the irr mutant strain (Table I). This observation is interesting, because recent studies demonstrate vicR is essential for growth in Streptococcus pneumoniae and can be cross-phosphorylated by a heterologous histidine kinase sensor protein (18). Thus, it is possible that an increase in vicR compensates for the lack of irr, resulting in similar growth rates in the wild-type and mutant strains (Fig. 1C). These data support the notion that there is regulatory cross-talk between two-component gene regulatory systems in bacterial pathogens.



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 2. Ihk-Irr regulates global gene expression patterns in GAS. A, Genes up- or down-regulated in the irr mutant GAS strain compared with the wild-type strain were identified by GAS microarrays and assigned to categories. Data for genes whose expression was significantly different between each strain at early exponential phase (OD600 = 0.35) and/or late exponential phase (OD600 = 0.75) of growth were combined for this analysis. B, Model schematic for Ihk-Irr-regulated detoxification of hydrogen peroxide. Transcript levels for genes increased (red boxes) or decreased (green boxes) in the irr mutant GAS strain compared with the wild-type strain identified by the microarray studies. White box with gor (glutathione reductase, SPy0813) indicates the gene was present but not differentially expressed. The model schematic is based on function of putative homolog encoded by the indicated genes.

 

View this table:
[in this window]
[in a new window]
  		Table I. Genes regulated by Irr-Ihka

 
Our finding that Ihk-Irr was important for GAS pathogenesis (Fig. 1) suggests that this transcription regulatory system controls expression of genes associated with virulence. However, only four genes encoding known virulence-associated factors, fbp (putative fibronectin-binding protein-like protein A, SPy1013), mf and mf3 (DNases), and sagA (streptolysin S, SPy0738) (19), were down-regulated in the irr mutant strain (Table I). Inasmuch as there are many fibronectin-binding proteins produced by GAS (20, 21, 22, 23, 24, 25), and the role of DNases in streptococcal pathogenesis is unclear (26, 27), attenuated virulence in the irr mutant strain is not readily explained by decreased fbp, mf, and mf3 transcript levels. In contrast, sagA is associated with increased tissue necrosis in mouse models of GAS infection (19, 28, 29). The observation that sagA transcript levels are decreased in the irr mutant strain correlates with reduced abscess size in infected mice (Fig. 1). Because previous studies indicate sagA expression is regulated by CsrR/S (30), modulation of sagA by Ihk-Irr may be indirect or, alternatively, controlled by multiple gene regulatory systems. Regardless, these data indicate that, in contrast to CsrR/S (31, 32) or Mga (31), Ihk-Irr has limited influence on expression of known GAS virulence factors.

Ihk-Irr controls expression of genes involved in cell wall formation

GAS genes encoding proteins involved in cell wall formation are up-regulated concomitantly with ihk and irr during neutrophil phagocytosis (7). Consistent with those findings, genes important in cell wall and/or peptidoglycan synthesis, including gidB (glucose-inhibited division protein, SPy0329), Spy0510, putative UDP-N-acetylmuramyl tripeptide synthetase (Spy1035), D-alanyl-D-alanyl carboxypeptidase (SPy1093), UDP-N-acetylmuramoylpentapeptide-lysine-N-(6)-lanyltransferase (Spy1205), dltB (SPy1311), and mraY (undecaprenyl-phosphate-UDP-MurNAc-pentapeptide phospho-MurNAc pentapeptide transferase, SPy1662) were down-regulated in the irr mutant GAS strain (Table I). These data suggest that Ihk-Irr regulates cell wall synthesis, a finding most compatible with the observation that the irr mutant strain is rapidly destroyed following neutrophil phagocytosis (7). Because neutrophil-derived cationic antimicrobial peptides such as {alpha}-defensin-1, LL-37, and cathepsin G kill bacteria by disrupting the cell envelope (cell wall and/or membrane) (33, 34, 35, 36, 37), reduced levels of genes involved in cell wall synthesis likely increase susceptibility of the irr mutant GAS strain to these peptides. This hypothesis is tested below.

Ihk-Irr regulates expression of oxidative stress genes and protects against killing by H2O2

Inasmuch as GAS elicits production of neutrophil ROS during phagocytosis (1, 7), the ability to endure neutrophil-derived ROS likely contributes to pathogen survival after phagocytosis. Therefore, one of our prominent findings was that Ihk-Irr regulates expression of genes encoding proteins that control cell redox status and protect against oxidative stress (38, 39). GAS genes encoding trx (thioredoxin, SPy1835), thioredoxin reductase (SPy0850), NADH peroxidase (SPy1681), nrdH (glutaredoxin, SPy1374), and ribonucleotide reductase (SPy1375) were down-regulated in the irr mutant strain (Table I). In contrast, only one gene involved in oxidative stress response, bsaA (SPy0605), which encodes glutathione peroxidase, was up-regulated (Table I). These data indicate that at least four possible biochemical pathways that detoxify ROS are down-regulated (Fig. 2B).

To determine whether down-regulation of genes involved in responses to oxidative stress reflects increased killing by ROS, we measured susceptibility of the wild-type and irr mutant GAS strains to H2O2 (Fig. 3A). There was concentration-dependent killing of both strains by H2O2 (Fig. 3A). However, the irr mutant strain was significantly more susceptible to H2O2-mediated killing than the wild-type strain (p < 0.016) (Fig. 3A). Although these findings suggest that Ihk-Irr protects GAS from H2O2-mediated killing by influencing expression of genes involved in the detoxification of ROS, it is also possible that increased killing of the irr mutant strain was due in part to altered cell wall synthesis.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 3. Ihk-Irr protects against killing by H2O2 and neutrophil primary granule components. A, Killing of wild-type (blue bars) and irr mutant (red bars) GAS strains by H2O2. Results are the mean ± SEM of 7–12 separate experiments. *, p < 0.016 vs irr mutant strain. B, Killing of wild-type and irr mutant GAS strains by solubilized neutrophil primary granule ({alpha}-granule) components. Results are the mean ± SEM of three to eight separate experiments. *, p < 0.009 vs irr mutant strain; **, p < 0.02 vs irr mutant strain. CE, Cell equivalents. C, Killing of wild-type and irr mutant strains by neutrophil antibacterial peptides. Results are the mean ± SEM of two to three experiments. *, p < 0.03 vs irr mutant strain.

 
Ihk-Irr facilitates resistance to killing by neutrophil granule components

In addition to eliminating pathogens with ROS, human neutrophils kill Gram-positive bacteria with antimicrobial peptides that disrupt cell envelope integrity (33, 34, 35, 36, 37, 40, 41). Therefore, we exposed wild-type and irr mutant GAS strains to human neutrophil granule fractions to determine whether there were differences in survival, thus reflecting altered cell wall synthesis. Neither strain was killed by neutrophil secondary granule components (data not shown). This finding is consistent with the idea that peptides and enzymes of secondary granules, such as hCAP-18 (42), are inactive zymogens that require proteolytic processing to obtain activity (43). In contrast, both strains were killed to various degrees by solubilized neutrophil primary granules, which contain active {alpha}-defensins, elastase, and cathepsin G (42) (Fig. 3B). Notably, the irr mutant strain was significantly more susceptible to killing by primary granule components compared with the wild-type strain (p < 0.009) (Fig. 3B). We next tested the streptocidal activity of LL-37 (44) (the active peptide form of hCAP-18), {alpha}-defensin-1, neutrophil elastase, and neutrophil cathepsin G to determine whether differences in survival noted between the irr mutant and wild-type strains could be attributed to a single granule component (Fig. 3C). Although each of the strains was killed by relatively low concentrations of these antibacterial peptides (Fig. 3C), significantly more of the irr mutant strain was killed by LL-37 and cathepsin G, each of which targets the cell envelope (37, 41). These results suggest that Ihk-Irr protects GAS from antibacterial neutrophil peptides by regulating cell wall synthesis and/or turnover.

ihk and irr are induced by H2O2 and neutrophil primary granules

To further understand the role of Ihk-Irr in pathogenesis, we exposed wild-type GAS to amounts of H2O2 representative of that within neutrophil phagosomes and analyzed ihk and irr transcript levels with TaqMan real-time PCR (Fig. 4). Expression of ihk and irr increased following exposure to H2O2, although the magnitude of change compared with untreated GAS was relatively moderate (≤2.0-fold at 30 min) (Fig. 4A). These results are consistent with the finding that ihk-irr is up-regulated during neutrophil phagocytosis and accompanying production of ROS (7).



View larger version (44K):
[in this window]
[in a new window]
  		FIGURE 4. ihk and irr are induced by H2O2 and human neutrophil primary granules. A, Induction of ihk and irr in the wild-type strain by 1 mM H2O2. Results are the mean ± SD fold-change of triplicate wells from a representative TaqMan experiment performed at least twice for each time point. B, Change in irr transcript levels after incubation with human neutrophil primary granule fractions ({alpha}), heat-inactivated neutrophil primary granule fractions ({Delta}{alpha}), neutrophil secondary granule fractions ({beta}), and heat-inactivated neutrophil secondary granule fractions ({Delta}{beta}). The data for induction of irr by H2O2 was included on the same scale for comparison (see dotted line). Results are the mean ± SD fold-change of triplicate wells from a representative TaqMan experiment performed twice. C, Time-resolved change in ihk and irr transcript following exposure of the wild-type GAS strain to neutrophil primary granules. Results are the mean ± SD fold-change of triplicate wells from a representative TaqMan experiment performed at least twice for each time point. D and E, Killing of serotype M1 and M18 GAS strains by 1 mM H2O2 (D) or solubilized human neutrophil {alpha}-granule components (E). Percent GAS survival is the mean ± SEM of six to seven separate experiments relative to untreated bacteria ({blacksquare}). F, Change in irr and ihk transcript levels in serotype M1 and M18 GAS strains after a 30-min incubation with 1 mM H2O2 or 107 cell equivalents of human neutrophil {alpha}-granules as indicated. Results are the mean ± SEM fold-change of three separate TaqMan experiments.

 
We next exposed GAS to purified neutrophil primary and secondary granule fractions and measured transcript levels of irr to determine whether expression of the gene is altered by these antibacterial components (Fig. 4B). Unexpectedly, exposure of the pathogen to neutrophil primary granules caused a robust increase in irr transcript compared with untreated bacteria (~22-fold at 30 min) (Fig. 4B). Neither secondary granules nor heat-inactivated granules altered irr transcript levels to the same extent (Fig. 4B). These findings indicate that increases in irr transcript were not simply due to the binding of primary granule components to the bacteria cell surface. Rather, the data suggest that increased irr expression is a pathogen response to cell wall and/or membrane damage caused by antibacterial peptides contained within neutrophil granules. Importantly, ihk and irr were each induced by neutrophil primary granule components in a time-dependent manner and in strains of GAS representative of those that cause invasive (serotype M1) and noninvasive (serotype M18) disease in humans (Fig. 4, C–F). Inasmuch as Ihk-Irr regulates expression of genes important for GAS survival in the host (Fig. 1 and Table I), these findings provide strong support to the idea that Ihk-Irr triggers pathogen-protective responses to innate host defense.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our understanding of how bacterial pathogens avert neutrophil killing following phagocytosis is limited. GAS alters patterns of global gene transcription during phagocytic interaction with neutrophils and resists the effects of neutrophil microbicidal components to survive and cause neutrophil necrosis (1, 6, 7, 8, 9, 10). The finding that GAS lacking functional Ihk-Irr are rapidly destroyed after phagocytosis suggests that this gene regulatory system may play a role in these processes (7). Moreover, irr is highly expressed in several GAS M-serotypes isolated from patients with pharyngitis, implying that Ihk-Irr is important for GAS pathogenesis in humans (7). However, none of those studies address a mechanism for triggering pathogen-protective responses nor is the molecular basis for prolonged survival within neutrophils known.

To that end, we investigated the role of Ihk-Irr in streptococcal pathogenesis and found that inactivation of irr attenuated virulence significantly, resulting in reduced soft tissue infection and more rapid resolution of bacteremia. Microarray analyses indicated that Ihk-Irr influenced expression of genes that regulate cell redox status and cell wall synthesis, and GAS lacking irr were rapidly killed by antibacterial peptides that target the cell envelope. These observations provide strong support to the idea that regulation of cell wall synthesis is critical for GAS pathogenesis. Recent studies have hinted that bacteria cell surface modification may be involved in averting destruction by neutrophils (10, 33). Resistance of microbes to the effects of neutrophil microbicidal components through bacteria cell wall synthesis or surface modification is thus an emerging theme in bacterial pathogenesis.

Based on our findings, we propose a model in which neutrophil-derived ROS and damage resulting from neutrophil antibacterial peptides activates/induces Ihk-Irr, which in turn alters GAS gene expression (Fig. 5). Induction of Ihk-Irr-regulated genes, especially those involved in cell wall synthesis, extends GAS survival such that neutrophil lysis occurs before the pathogen is eliminated (1), thus promoting dissemination and disease. Consistent with our model for the pathogen survival response, the PhoP-PhoQ and PmrA-PmrB two-component gene regulatory systems of Salmonella typhimurium and Pseudomonas aeruginosa are activated by cationic antimicrobial peptides, and in turn, regulate resistance to antimicrobial peptides (45, 46, 47, 48). Activation of PhoP-PhoQ in Salmonella by antimicrobial peptides alters global gene and protein expression patterns (45), a process likely similar for Ihk-Irr of GAS.



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 5. A model for the Ihk-Irr-mediated pathogen survival response. irr and ihk are each induced by ROS and primary granule proteins during phagocytosis. Based on the microarray results, Ihk-Irr influences expression of genes comprising the survival response, including those involved in redox status and cell wall synthesis. Induction of these genes prolongs pathogen survival, resulting in neutrophil lysis and GAS dissemination.

 
Inasmuch as neutrophils are the most prominent innate immune effector cells in humans, the host factors that induce the pathogen survival response described in this study are prevalent in nearly all bacterial infections. The process for triggering microbe survival responses is thus a general mechanism used by pathogens to detect and evade human innate immunity. A better understanding of the host-pathogen interface at both the cell and molecular levels is critical to our understanding, treatment, and control of human diseases caused by bacteria. Importantly, our studies identify specific genes in the bacterial pathogen survival response as potential targets to control human infections.


   Acknowledgments
 
We thank J. Scott for GAS strains JRS4 and JRS550, and A. Mora for assistance with photography.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Frank R. DeLeo, Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 S. 4th Street, Hamilton, MT 59840. E-mail address: fdeleo{at}niaid.nih.gov Back

2 Abbreviations used in this paper: ROS, reactive oxygen species; GAS, group A Streptococcus or Streptococcus pyogenes; irr mutant, irr-negative mutant; TSA, tyramide signal amplification; ORF, open reading frame. Back

3 The on-line version of this article contains supplemental material. Back

Received for publication February 10, 2004. Accepted for publication May 11, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kobayashi, S. D., K. R. Braughton, A. R. Whitney, J. M. Voyich, T. G. Schwan, J. M. Musser, F. R. DeLeo. 2003. Bacterial pathogens modulate an apoptosis differentiation program in human neutrophils. Proc. Natl. Acad. Sci. USA 100:10948.[Abstract/Free Full Text]
  2. Yoshiie, K., H. Y. Kim, J. Mott, Y. Rikihisa. 2000. Intracellular infection by the human granulocytic ehrlichiosis agent inhibits neutrophil apoptosis. Infect. Immun. 68:1125.[Abstract/Free Full Text]
  3. Scaife, H., Z. Woldehiwet, C. A. Hart, S. W. Edwards. 2003. Anaplasma phagocytophilum reduces neutrophil apoptosis in vitro. Infect. Immun. 71:1995.[Abstract/Free Full Text]
  4. Lei, B., F. R. DeLeo, N. P. Hoe, M. R. Graham, S. M. Mackie, R. L. Cole, M. Liu, H. R. Hill, D. E. Low, M. J. Federle, et al 2001. Evasion of human innate and acquired immunity by a bacterial homolog of CD11b that inhibits opsonophagocytosis. Nat. Med. 7:1298.[Medline]
  5. Hoe, N. P., R. M. Ireland, F. R. DeLeo, B. G. Gowen, D. W. Dorward, J. M. Voyich, M. Liu, E. H. Burns, Jr, D. M. Culnan, A. Bretscher, J. M. Musser. 2002. Insight into the molecular basis of pathogen abundance: group A Streptococcus inhibitor of complement inhibits bacterial adherence and internalization into human cells. Proc. Natl. Acad. Sci. USA 99:7646.[Abstract/Free Full Text]
  6. Collin, M., M. D. Svensson, A. G. Sjoholm, J. C. Jensenius, U. Sjobring, A. Olsen. 2002. EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis. Infect. Immun. 70:6646.[Abstract/Free Full Text]
  7. Voyich, J. M., D. E. Sturdevant, K. R. Braughton, S. D. Kobayashi, B. Lei, K. Virtaneva, D. Dorward, J. M. Musser, F. R. DeLeo. 2003. Genome-wide protective response used by group A Streptococcus to evade destruction by human polymorphonuclear leukocytes. Proc. Natl. Acad. Sci. USA 100:1996.[Abstract/Free Full Text]
  8. Staali, L., M. Morgelin, L. Bjorck, H. Tapper. 2003. Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils. Cell. Microbiol. 5:253.[Medline]
  9. Medina, E., O. Goldmann, A. W. Toppel, G. S. Chhatwal. 2003. Survival of Streptococcus pyogenes within host phagocytic cells: a pathogenic mechanism for persistence and systemic invasion. J. Infect. Dis. 187:597.[Medline]
  10. Medina, E. O., M. Rohde, G. S. Chhatwal. 2003. Intracellular survival of Streptococcus pyogenes in polymorphonuclear cells results in increased bacterial virulence. Infect. Immun. 71:5376.[Abstract/Free Full Text]
  11. Federle, M. J., K. S. McIver, J. R. Scott. 1999. A response regulator that represses transcription of several virulence operons in the group A Streptococcus. J. Bacteriol. 181:3649.[Abstract/Free Full Text]
  12. Ferretti, J. J., W. M. McShan, D. Ajdic, D. J. Savic, G. Savic, K. Lyon, C. Primeaux, S. Sezate, A. N. Suvorov, S. Kenton, et al 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658.[Abstract/Free Full Text]
  13. Smoot, J. C., K. D. Barbian, J. J. Van Gompel, L. M. Smoot, M. S. Chaussee, G. L. Sylva, D. E. Sturdevant, S. M. Ricklefs, S. F. Porcella, L. D. Parkins, et al 2002. Genome sequence and comparative microarray analysis of serotype M18 group A Streptococcus strains associated with acute rheumatic fever outbreaks. Proc. Natl. Acad. Sci. USA 99:4668.[Abstract/Free Full Text]
  14. Boyum, A.. 1968. Isolation of mononuclear cells and granulocytes from human blood: isolation of mononuclear cells by one centrifugation, and of granulocytes by combing centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl. 97:77.[Medline]
  15. Beres, S. B., G. L. Sylva, K. D. Barbian, B. Lei, J. S. Hoff, N. D. Mammarella, M. Y. Liu, J. C. Smoot, S. F. Porcella, L. D. Parkins, et al 2002. Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence. Proc. Natl. Acad. Sci. USA 99:10078.[Abstract/Free Full Text]
  16. Borregaard, N., J. M. Heiple, E. R. Simons, R. A. Clark. 1983. Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation. J. Cell Biol. 97:52.[Abstract/Free Full Text]
  17. Biswas, I., J. B. Scott. 2003. Identification of rocA, a positive regulator of covR expression in the group A Streptococcus. J. Bacteriol. 185:3081.[Abstract/Free Full Text]
  18. Wagner, C., A. Ad Saizieu, H. J. Schonfeld, M. Kamber, R. Lange, C. J. Thompson, M. G. Page. 2002. Genetic analysis and functional characterization of the Streptococcus pneumoniae vic operon. Infect. Immun. 70:6121.[Abstract/Free Full Text]
  19. Betschel, S. D., S. M. Borgia, N. L. Barg, D. E. Low, J. C. S. De Azavedo. 1998. Reduced virulence of group A streptococcal TN916 mutants that do not produce streptolysin S. Infect. Immun. 66:1671.[Abstract/Free Full Text]
  20. Hanski, E., M. Caparon. 1992. Protein F, a fibronectin-binding protein, is an adhesin of the group A streptococcus Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 89:6172.[Abstract/Free Full Text]
  21. Talay, S. R., P. Valentin-Weigand, P. G. Jerlstrom, K. N. Timmis, G. S. Chhatwal. 1992. Fibronectin-binding protein of Streptococcus pyogenes: sequence of the binding domain involved in adherence of streptococci to epithelial cells. Infect. Immun. 60:3837.[Abstract/Free Full Text]
  22. Courtney, H. S., Y. Li, J. B. Dale, D. L. Hasty. 1994. Cloning, sequencing, and expression of a fibronectin/fibrinogen-binding protein from group A streptococci. Infect. Immun. 62:3937.[Abstract/Free Full Text]
  23. Rocha, C. L., V. A. Fischetti. 1999. Identification and characterization of a novel fibronectin-binding protein on the surface of group A streptococci. Infect. Immun. 67:2720.[Abstract/Free Full Text]
  24. Terao, Y., S. Kawabata, E. Kunitomo, J. Murakami, I. Nakagawa, S. Hamada. 2001. Fba, a novel fibronectin-binding protein from Streptococcus pyogenes, promotes bacterial entry into epithelial cells, and the fba gene is positively transcribed under the Mga regulator. Mol. Microbiol. 42:75.[Medline]
  25. Terao, Y., S. Kawabata, M. Nakata, I. Nakagawa, S. Hamada. 2002. Molecular characterization of a novel fibronectin-binding protein of Streptococcus pyogenes strains isolated from toxic shock-like syndrome patients. J. Biol. Chem. 277:47428.[Abstract/Free Full Text]
  26. Podbielski, A., I. Zarges, A. Flosdorff, J. Weber-Heynemann. 1996. Molecular characterization of a major serotype M49 group A streptococcal DNase gene (sdaD). Infect. Immun. 64:5349.[Abstract]
  27. Sriskandan, S., M. Unnikrishnan, T. Krausz, J. Cohen. Mitogenic factor (MF) is the major DNase of serotype M89 Streptococcus pyogenes. Microbiology 146:2785.
  28. Humar, D., V. Datta, D. J. Bast, B. Beall, J. C. De Azavedo, V. Nizet. 2002. Streptolysin S and necrotising infections produced by group G streptococcus. Lancet 359:124.[Medline]
  29. Engleberg, N. C., A. Heath, K. Vardaman, V. J. DiRita. 2004. Contribution of CsrR-regulated virulence factors to the progress and outcome of murine skin infections by Streptococcus pyogenes. Infect. Immun. 72:623.[Abstract/Free Full Text]
  30. Heath, A., V. J. DiRita, N. L. Barg, N. C. Engleberg. 1999. A two-component regulatory system, CsrR-CsrS, represses expression of three Streptococcus pyogenes virulence factors, hyaluronic acid capsule, streptolysin S, and pyrogenic exotoxin B. Infect. Immun. 67:5298.[Abstract/Free Full Text]
  31. Kreikemeyer, B., K. S. McIver, A. Podbielski. 2003. Virulence factor regulation and regulatory networks in Streptococcus pyogenes and their impact on pathogen-host interactions. Trends Microbiol. 11:224.[Medline]
  32. Graham, M. R., L. M. Smoot, C. A. Migliaccio, K. Virtaneva, D. E. Sturdevant, S. F. Porcella, M. J. Federle, G. J. Adams, J. R. Scott, J. M. Musser. 2002. Virulence control in group A Streptococcus by a two-component gene regulatory system: global expression profiling and in vivo infection modeling. Proc. Natl. Acad. Sci. USA 99:13855.[Abstract/Free Full Text]
  33. Poyart, C., E. Pellegrini, M. Marceau, M. Baptista, F. Jaubert, M. C. Lamy, P. Trieu-Cuot. 2003. Attenuated virulence of Streptococcus agalactiae deficient in D-alanyl-lipoteichoic acid is due to an increased susceptibility to defensins and phagocytic cells. Mol. Microbiol. 49:1615.[Medline]
  34. Peschel, A., M. Otto, R. W. Jack, H. Kalbacher, G. Jung, F. Götz. 1999. Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. J. Biol. Chem. 274:8405.[Abstract/Free Full Text]
  35. Ganz, T., M. E. Selsted, D. Szklarek, S. S. Harwig, K. Daher, D. F. Bainton, R. I. Lehrer. 1985. Defensins: natural peptide antibiotics of human neutrophils. J. Clin. Invest. 76:1427.
  36. Agerberth, B., H. Gunne, J. Odeberg, P. Kogner, H. G. Boman, G. H. Gudmundsson. 1995. FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc. Natl. Acad. Sci. USA 92:195.[Abstract/Free Full Text]
  37. Thorne, K. J., R. C. Oliver, A. J. Barrett. 1976. Lysis and killing of bacteria by lysosomal proteinases. Infect. Immun. 14:555.[Abstract/Free Full Text]
  38. Ritz, D., H. Patel, B. Doan, M. Zheng, F. Aslund, G. Storz, J. Beckwith. 2000. Thioredoxin 2 is involved in the oxidated stress response in Escherichia coli. J. Biol. Chem. 275:2505.[Abstract/Free Full Text]
  39. Nishinaka, Y., H. Masutani, H. Nakamura, J. Yodoi. 2001. Regulatory roles of thioredoxin in oxidative stress-induced cellular responses. Redox Rep. 6:289.[Medline]
  40. Lehrer, R. I., A. Barton, K. A. Daher, S. S. Harwig, T. Ganz, M. E. Selsted. 1989. Interaction of human defensins with Escherichia coli: mechanism of bactericidal activity. J. Clin. Invest. 84:553.
  41. Shafer, W. M., V. C. Onunka, L. E. Martin. 1986. Antigonococcal activity of human neutrophil cathepsin G. Infect. Immun. 54:184.[Abstract/Free Full Text]
  42. Sorensen, O., K. Arnljots, J. B. Cowland, D. F. Bainton, N. Borregaard. 1997. The human antibacterial cathelicidin, hCAP-18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood 90:2796.[Abstract/Free Full Text]
  43. Borregaard, N., J. B. Cowland. 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89:3503.[Free Full Text]
  44. Nizet, V., T. Ohtake, X. Lauth, J. Trowbridge, J. Rudisill, R. A. Dorschner, V. Pestonjamasp, J. Piraino, K. Huttner, R. L. Gallo. 2001. Innate antimicrobial peptide protects the skin from invasive bacterial infection. Nature 22:454.
  45. Bader, M. W., W. W. Navarre, W. Shiau, H. Nikaido, J. G. Frye, M. McClelland, F. C. Fang, S. I. Miller. 2003. Regulation of Salmonella typhimurium virulence gene expression by cationic antimicrobial peptides. Mol. Microbiol. 50:219.[Medline]
  46. Macfarlane, E. L., A. Kwasnicka, M. M. Ochs, R. E. Hancock. 1999. PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol. Microbiol. 34:305.[Medline]
  47. Macfarlane, E. L., A. Kwasnicka, R. E. Hancock. 2000. Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides. Microbiology 146:2543.[Abstract/Free Full Text]
  48. McPhee, J. B., S. Lewenza, R. E. Hancock. 2003. Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol. Microbiol. 50:205.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. M. Palazzolo-Ballance, M. L. Reniere, K. R. Braughton, D. E. Sturdevant, M. Otto, B. N. Kreiswirth, E. P. Skaar, and F. R. DeLeo
Neutrophil Microbicides Induce a Pathogen Survival Response in Community-Associated Methicillin-Resistant Staphylococcus aureus
J. Immunol., January 1, 2008; 180(1): 500 - 509.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
M. R. Graham, K. Virtaneva, S. F. Porcella, D. J. Gardner, R. D. Long, D. M. Welty, W. T. Barry, C. A. Johnson, L. D. Parkins, F. A. Wright, et al.
Analysis of the Transcriptome of Group A Streptococcus in Mouse Soft Tissue Infection
Am. J. Pathol., September 1, 2006; 169(3): 927 - 942.
[Abstract] [Full Text] [PDF]

Home page
 MicrobiologyHome page
M. Liu, T. S. Hanks, J. Zhang, M. J. McClure, D. W. Siemsen, J. L. Elser, M. T. Quinn, and B. Lei
Defects in ex vivo and in vivo growth and sensitivity to osmotic stress of group A Streptococcus caused by interruption of response regulator gene vicR.
Microbiology, April 1, 2006; 152(Pt 4): 967 - 978.
[Abstract] [Full Text] [PDF]

Home page
 Infect. Immun.Home page
I. Sitkiewicz and J. M. Musser
Expression Microarray and Mouse Virulence Analysis of Four Conserved Two-Component Gene Regulatory Systems in Group A Streptococcus
Infect. Immun., February 1, 2006; 74(2): 1339 - 1351.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
J. M. Musser and F. R. DeLeo
Toward a Genome-Wide Systems Biology Analysis of Host-Pathogen Interactions in Group A Streptococcus
Am. J. Pathol., December 1, 2005; 167(6): 1461 - 1472.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. K. Femling, W. M. Nauseef, and J. P. Weiss
Synergy between Extracellular Group IIA Phospholipase A2 and Phagocyte NADPH Oxidase in Digestion of Phospholipids of Staphylococcus aureus Ingested by Human Neutrophils
J. Immunol., October 1, 2005; 175(7): 4653 - 4661.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. M. Voyich, K. R. Braughton, D. E. Sturdevant, A. R. Whitney, B. Said-Salim, S. F. Porcella, R. D. Long, D. W. Dorward, D. J. Gardner, B. N. Kreiswirth, et al.
Insights into Mechanisms Used by Staphylococcus aureus to Avoid Destruction by Human Neutrophils
J. Immunol., September 15, 2005; 175(6): 3907 - 3919.
[Abstract] [Full Text] [PDF]

Home page
 Appl. Environ. Microbiol.Home page
J. Letowski, A. Bravo, R. Brousseau, and L. Masson
Assessment of cry1 Gene Contents of Bacillus thuringiensis Strains by Use of DNA Microarrays
Appl. Envir. Microbiol., September 1, 2005; 71(9): 5391 - 5398.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
K. Virtaneva, S. F. Porcella, M. R. Graham, R. M. Ireland, C. A. Johnson, S. M. Ricklefs, I. Babar, L. D. Parkins, R. A. Romero, G. J. Corn, et al.
Longitudinal analysis of the group A Streptococcus transcriptome in experimental pharyngitis in cynomolgus macaques
PNAS, June 21, 2005; 102(25): 9014 - 9019.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
M. R. Graham, K. Virtaneva, S. F. Porcella, W. T. Barry, B. B. Gowen, C. R. Johnson, F. A. Wright, and J. M. Musser
Group A Streptococcus Transcriptome Dynamics during Growth in Human Blood Reveals Bacterial Adaptive and Survival Strategies
Am. J. Pathol., February 1, 2005; 166(2): 455 - 465.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Voyich, J. M.
Right arrow Articles by DeLeo, F. R.
Right arrow Search for Related Content
PubMed