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1.  Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils 
PLoS ONE  2012;7(2):e31524.
The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC).
In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-α, IL-1-α/β), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages.
This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations.
PMCID: PMC3279406  PMID: 22348096
2.  Functional Analysis of the Quorum-Sensing Streptococcal Invasion Locus (sil) 
PLoS Pathogens  2009;5(11):e1000651.
Group A streptococcus (GAS) causes a wide variety of human diseases, and at the same time, GAS can also circulate without producing symptoms, similar to its close commensal relative, group G streptococcus (GGS). We previously identified, by transposon-tagged mutagenesis, the streptococcal invasion locus (sil). sil is a quorum-sensing regulated locus which is activated by the autoinducer peptide SilCR through the two-component system SilA-SilB. Here we characterize the DNA promoter region necessary for SilA-mediated activation. This site is composed of two direct repeats of 10 bp, separated by a spacer of 11 bp. Fusion of this site to gfp allowed us to systematically introduce single-base substitutions in the repeats region and to assess the relative contribution of various positions to promoter strength. We then developed an algorithm giving different weights to these positions, and performed a chromosome-wide bioinformatics search which was validated by transcriptome analysis. We identified 13 genes, mostly bacteriocin related, that are directly under the control of SilA. Having developed the ability to quantify SilCR signaling via GFP accumulation prompted us to search for GAS and GGS strains that sense and produce SilCR. While the majority of GAS strains lost sil, all GGS strains examined still possess the locus and ∼63% are able to respond to exogenously added SilCR. By triggering the autoinduction circle using a minute concentration of synthetic SilCR, we identified GAS and GGS strains that are capable of sensing and naturally producing SilCR, and showed that SilCR can be sensed across these streptococci species. These findings suggest that sil may be involved in colonization and establishment of commensal host-bacterial relationships.
Author Summary
Cell-to-cell communication in bacteria is termed quorum-sensing (QS), which is triggered by signaling molecules called autoinducers. In streptococci, autoinducers are synthesized as immature peptides that are processed, secreted, and then sensed by two-component systems (TCSs). As a result, the autoinducer's own expression is upregulated (autoinduction), subsequently creating an ultrasensitive switch that turns on more genes. Group A streptococcus (GAS) is a human pathogen that causes many infections, including necrotizing fasciitis (NF). Previously, we identified in a NF GAS strain a QS locus termed streptococcal invasion locus (sil). Due to a mutation in the autoinducer peptide-SilCR, it is not produced by this strain. Here we sought to better explore sil and to examine if SilCR can be produced by other GAS strains, or strains of its close relative group G streptococcus (GGS). To this end, we characterized the DNA promoter region responsible for the TCS-mediated activation upon sensing of SilCR, and based on bioinformatics and transcriptome analyses we identified genes that are directly affected by the autoinducer peptide. By converting SilCR response to fluorescence production and turning on the autoinduction circle with minute concentrations of synthetic SilCR, we discovered naturally SilCR-producing GAS and GGS strains, and showed that SilCR can be sensed across these species. Our study describes a novel way of cell-to-cell communications among streptococci.
PMCID: PMC2766830  PMID: 19893632
3.  The IL-8 Protease SpyCEP/ScpC of Group A Streptococcus Promotes Resistance to Neutrophil Killing 
Cell host & microbe  2008;4(2):170-178.
Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (Cepl) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.
PMCID: PMC2631432  PMID: 18692776

Results 1-3 (3)