Mycobacterium avium Biofilm Attenuates Mononuclear Phagocyte Function by Triggering Hyperstimulation and Apoptosis during Early Infection

ABSTRACTMycobacterium aviumsubsp.hominissuisis an opportunistic human pathogen that has been shown to form biofilmin vitroandin vivo. Biofilm formationin vivoappears to be associated with infections in the respiratory tract of the host. The reasoning behind howM. aviumsubsp.hominissuisbiofilm is allowed to establish and persist without being cleared by the innate immune system is currently unknown. To identify the mechanism responsible for this, we developed anin vitromodel using THP-1 human mononuclear phagocytes cocultured with establishedM. aviumsubsp.hominissuisbiofilm and surveyed various aspects of the interaction, including phagocyte stimulation and response, bacterial killing, and apoptosis.M. aviumsubsp.hominissuisbiofilm triggered robust tumor necrosis factor alpha (TNF-α) release from THP-1 cells as well as superoxide and nitric oxide production. Surprisingly, the hyperstimulated phagocytes did not effectively eliminate the cells of the biofilm, even when prestimulated with gamma interferon (IFN-γ) or TNF-α or cocultured with natural killer cells (which have been shown to induce anti-M. aviumsubsp.hominissuisactivity when added to THP-1 cells infected with planktonicM. aviumsubsp.hominissuis). Time-lapse microscopy and the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay determined that contact with theM. aviumsubsp.hominissuisbiofilm led to early, widespread onset of apoptosis, which is not seen until much later in planktonicM. aviumsubsp.hominissuisinfection. Blocking TNF-α or TNF-R1 during interaction with the biofilm significantly reduced THP-1 apoptosis but did not lead to elimination ofM. aviumsubsp.hominissuis. Our data collectively indicate thatM. aviumsubsp.hominissuisbiofilm induces TNF-α-driven hyperstimulation and apoptosis of surveilling phagocytes, which prevents clearance of the biofilm by cells of the innate immune system and allows the biofilm-associated infection to persist.

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Interaction of Antibiotics with Innate Host Defense Factors against Salmonella enterica Serotype Newport

mSphere ◽

10.1128/msphere.00410-17 ◽

2017 ◽

Vol 2 (6) ◽

Cited By ~ 15

Author(s):

George Sakoulas ◽

Monika Kumaraswamy ◽

Armin Kousha ◽

Victor Nizet

Keyword(s):

Innate Immunity ◽

Immune System ◽

Innate Immune System ◽

Salmonella Enterica ◽

Clinical Performance ◽

Innate Immune ◽

Content Type ◽

Antimicrobial Assay

ABSTRACT It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo. This study examines the pharmacodynamics of antimicrobials that are used to treat Salmonella with each other and with key components of the innate immune system. Antimicrobial synergy was assessed using time-kill and checkerboard assays. Antimicrobial interactions with innate immunity were studied by employing cathelicidin LL-37, whole-blood, and neutrophil killing assays. Ceftriaxone and ciprofloxacin were found to be synergistic in vitro against Salmonella enterica serotype Newport. Ceftriaxone, ciprofloxacin, and azithromycin each demonstrated synergy with the human cathelicidin defense peptide LL-37 in killing Salmonella. Exposure of Salmonella to sub-MICs of ceftriaxone resulted in enhanced susceptibility to LL-37, whole blood, and neutrophil killing. The activity of antibiotics in vivo against Salmonella may be underestimated in bacteriologic media lacking components of innate immunity. The pharmacodynamic interactions of antibiotics used to treat Salmonella with each other and with components of innate immunity warrant further study in light of recent findings showing in vivo selection of antimicrobial resistance by single agents in this pathogen. IMPORTANCE It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo.

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Redundant Catalases Detoxify Phagocyte Reactive Oxygen and Facilitate Histoplasma capsulatum Pathogenesis

Infection and Immunity ◽

10.1128/iai.00173-13 ◽

2013 ◽

Vol 81 (7) ◽

pp. 2334-2346 ◽

Cited By ~ 35

Author(s):

Eric D. Holbrook ◽

Katherine A. Smolnycki ◽

Brian H. Youseff ◽

Chad A. Rappleye

Keyword(s):

Immune System ◽

Innate Immune System ◽

Histoplasma Capsulatum ◽

Reactive Oxygen ◽

Innate Immune ◽

Respiratory Pathogen ◽

Human Neutrophils ◽

Content Type

ABSTRACTHistoplasma capsulatumis a respiratory pathogen that infects phagocytic cells. The mechanisms allowingHistoplasmato overcome toxic reactive oxygen molecules produced by the innate immune system are an integral part ofHistoplasma's ability to survive during infection. To probe the contribution ofHistoplasmacatalases in oxidative stress defense, we created and analyzed the virulence defects of mutants lacking CatB and CatP, which are responsible for extracellular and intracellular catalase activities, respectively. Both CatB and CatP protectedHistoplasmafrom peroxide challengein vitroand from antimicrobial reactive oxygen produced by human neutrophils and activated macrophages. Optimal protection required both catalases, as the survival of a double mutant lacking both CatB and CatP was lower than that of single-catalase-deficient cells. Although CatB contributed to reactive oxygen species defensesin vitro, CatB was dispensable for lung infection and extrapulmonary disseminationin vivo. Loss of CatB from a strain also lacking superoxide dismutase (Sod3) did not further reduce the survival ofHistoplasmayeasts. Nevertheless, some catalase function was required for pathogenesis since simultaneous loss of both CatB and CatP attenuatedHistoplasmavirulencein vivo. These results demonstrate thatHistoplasma's dual catalases comprise a system that enablesHistoplasmato efficiently overcome the reactive oxygen produced by the innate immune system.

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Flagellar Motility Is a Key Determinant of the Magnitude of the Inflammasome Response to Pseudomonas aeruginosa

Infection and Immunity ◽

10.1128/iai.00054-13 ◽

2013 ◽

Vol 81 (6) ◽

pp. 2043-2052 ◽

Cited By ~ 40

Author(s):

Yash R. Patankar ◽

Rustin R. Lovewell ◽

Matthew E. Poynter ◽

Jeevan Jyot ◽

Barbara I. Kazmierczak ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Immune System ◽

Innate Immune System ◽

Innate Immune ◽

Bacterial Motility ◽

Inflammasome Activation ◽

Flagellar Motility ◽

Content Type

ABSTRACTWe previously demonstrated that bacterial flagellar motility is a fundamental mechanism by which host phagocytes bind and ingest bacteria. Correspondingly, loss of bacterial motility, consistently observed in clinical isolates from chronicPseudomonas aeruginosainfections, enables bacteria to evade association and ingestion ofP. aeruginosaby phagocytes bothin vitroandin vivo. Since bacterial interactions with the phagocyte cell surface are required for type three secretion system-dependent NLRC4 inflammasome activation byP. aeruginosa, we hypothesized that reduced bacterial association with phagocytes due to loss of bacterial motility, independent of flagellar expression, will lead to reduced inflammasome activation. Here we report that inflammasome activation is reduced in response to nonmotileP. aeruginosa. NonmotileP. aeruginosaelicits reduced IL-1β production as well as caspase-1 activation by peritoneal macrophages and bone marrow-derived dendritic cellsin vitro. Importantly, nonmotileP. aeruginosaalso elicits reduced IL-1β levelsin vivoin comparison to those elicited by wild-typeP. aeruginosa. This is the first demonstration that loss of bacterial motility results in reduced inflammasome activation and antibacterial IL-1β host response. These results provide a critical insight into how the innate immune system responds to bacterial motility and, correspondingly, how pathogens have evolved mechanisms to evade the innate immune system.

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Activation of the Innate Immune System by Treponema denticola Periplasmic Flagella through Toll-Like Receptor 2

Infection and Immunity ◽

10.1128/iai.00573-17 ◽

2017 ◽

Vol 86 (1) ◽

Cited By ~ 1

Author(s):

John Ruby ◽

Michael Martin ◽

Michael J. Passineau ◽

Valentina Godovikova ◽

J. Christopher Fenno ◽

...

Keyword(s):

Immune System ◽

Innate Immune System ◽

Innate Immune ◽

Treponema Denticola ◽

Toll Like Receptor ◽

Wild Type ◽

Content Type ◽

Tnf Α ◽

Toll Like Receptor 2 ◽

Periplasmic Flagella

ABSTRACTTreponema denticolais an indigenous oral spirochete that inhabits the gingival sulcus or periodontal pocket. Increased numbers of oral treponemes within this environment are associated with localized periodontal inflammation, and they are also part of an anaerobic polymicrobial consortium responsible for endodontic infections. Previous studies have indicated thatT. denticolastimulates the innate immune system through Toll-like receptor 2 (TLR2); however, the pathogen-associated molecular patterns (PAMPs) responsible forT. denticolaactivation of the innate immune system are currently not well defined. In this study, we investigated the role played byT. denticolaperiplasmic flagella (PF), unique motility organelles of spirochetes, in stimulating an innate immune response. Wild-typeT. denticolastimulated the production of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, IL-10, and IL-12 by monocytes from human peripheral blood mononuclear cells, while its isogenic nonmotile mutant lacking PF resulted in significantly diminished cytokine stimulation. In addition, highly purified PF were able to dose dependently stimulate cytokine TNF-α, IL-1β, IL-6, IL-10, and IL-12 production in human monocytes. Wild-typeT. denticolaand the purified PF triggered activation of NF-κB through TLR2, as determined using a variety of TLR-transfected human embryonic 293 cell lines, while the PF-deficient mutants lacked the ability to stimulate, and the complemented PF-positiveT. denticolastrain restored the activation. These findings suggest thatT. denticolastimulates the innate immune system in a TLR2-dependent fashion and that PF are a key bacterial component involved in this process.

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216 Anti-tumor activity of iosH2 by blocking LILRB2 receptor signalling

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0216 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A235-A235

Author(s):

Osiris Marroquin Belaunzaran ◽

Anahita Rafiei ◽

Anil Kumar ◽

Julia Kolibaba ◽

Lorenz Vogt ◽

...

Keyword(s):

T Cells ◽

Immune System ◽

T Cell ◽

Innate Immune System ◽

Cell Activation ◽

Innate Immune ◽

High Affinity ◽

Anti Tumor Activity

BackgroundThe human leukocyte immunoglobulin-like receptor family B (LILR B) acts as check point blockade of the innate immune system by inhibiting leukocyte activation through SHP phosphatase recruitment. Some of the physiological ligands include classical HLA class I molecules, including beta-2-microglobulin (B2M) free open conformers (OC). Natural HLA-OC expression is known from autoimmune disease leading to immune activation by pleiotropic effects since they bind to LILRB and KIR family members reducing Treg and MDSC numbers and increased effector T-cell and NK-cell activation, respectively. We have generated an IgG4-HLA-57 open conformer (OC) molecule (iosH2) with high affinity for LILRB molecules and demonstrate its anti-cancer activity in vitro and in vivo.Methods iosH2 was produced by transient gene expression in CHO cells and purified by standard chromatography. Affinity of iosH2 binding was quantified by ELISA and SPR analysis. HLA-G mediated signaling and competition was assessed using functional cell lines. Effect of iosH2 on activation of SHP1/2 was assessed using Western Blot. Functional assays including in vitro polarization and phagocytosis potential of primary macrophages was assessed by flow cytometry in the presence of iosH2 or isotype control. Effect of iosH2 on T cell activation was evaluated in co-cultures of cancer and T cells. Mouse models were used to assess in vivo activity.Results iosH2 binds to LILRB2 with high affinity and blocks the activation of HLA-G. In addition, iosH2 blocks receptor-mediated activation of SHP1/2. iosH2 promotes a shift from M2 to M1 macrophages with enhanced tumor cell phagocytosis in vitro. iosH2 enhances activation and killing potential of T cells in cancer cells and T cells co-culture assay. iosH2 exerts therapeutic efficacy in mouse transgenic (melanoma) and different syngeneic tumor models (e.g. pancreatic, colon and breast cancer) as monotherapy. Moreover, it acts synergistically in vivo with PD1 blocking antibodies achieving long-term tumor control. Ex vivo tumor sample analysis demonstrates a significant reduction of MDSC and Tregs and a shift towards an activated inflammatory M1 macrophage phenotype. Loss of MDSC functionality was paralleled by enhanced CD8+ T cell expansion and activity.Conclusions iosH2 binds to LILRB2 with high affinity, restores immune cell function in vitro and demonstrates anti-tumor activity in different in vivo mouse models. In addition, it acts synergistically in vivo with PD1. iosH2 is a first-in-class OC therapeutic with robust anti-tumor activity by promoting key components of the innate immune system. Clinical development is under way and phase I trial in preparation.

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A “Loss-of-Tolerance” Phenotype of the Liver Macrophage System to LPS Induces Overwhelming Systemic Inflammation and Early Death in a Knockout-Transgenic Mouse Model of Sickle Cell Disease.

Blood ◽

10.1182/blood.v106.11.2337.2337 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2337-2337

Author(s):

Steffen E. Meiler ◽

Ilka Theruvath ◽

Marlene Wade ◽

C. Alvin Head

Keyword(s):

Immune Response ◽

Immune System ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Innate Immune System ◽

Innate Immune ◽

Cell Disease ◽

Liver Macrophage ◽

Tnf Α

Abstract Infections and death from fulminant sepsis remain a constant threat to patients with sickle cell disease (SCD). Established explanations include an impaired ability to contain and eliminate pathogenic organisms due to defects in spleen function and adaptive immunity. To the contrary, very little is known about the “first-line” response of the innate immune system after contact with pathogen associated cell components. An escalated immune (inflammatory) response to microbial structures would provide an alternative mechanism to explain increased rates of infectious complications and septic death in sickle cell patients. To test this hypothesis, knockout-transgenic mice homozygous for the human β-sickle globin gene (SS) were treated with a low dose of the canonical infectious stimulus lipopolysaccharide (0.5 μg/g bw; i.p.; 22°C) and compared to heterozygous sickle trait (ST) and C57BL/6 animals. Phenotypically, sickle mice appeared much sicker after LPS and displayed strict seclusion behavior, cessation of food intake, and physiological signs of stress. Body core (rectal) temperature decreased precipitously and irreversibly in sickle animals (~12°C/8hrs vs. ~2°C/8hrs [ST and C57BL/6]) followed by rapid death (50%/12h; 100%/48h vs 0% [ST; C57BL/6]). Analysis of the LD50 demonstrated an ~500-fold increased sensitivity to LPS in sickle mice (0.05 μg/g vs 25 μg/g [ST]). Serum cytokines (TNF-α, IL-6) were dramatically up-regulated in SS mice compared to control (TNF-α: 16-fold/2 hr post LPS, 100-fold/3 hr post LPS). Organ-specific immunohistochemical analysis of the marker cytokine TNF-α in liver, bone marrow, spleen, lung, and kidney four hrs after LPS revealed an astonishingly super-induced expression in the liver of sickle animals compared to controls. The liver of sickle animals showed several areas of coagulative liver necrosis unrelated to LPS and consistent with ischemic injury from recurrent sickle-mediated vascular occlusion. Immunoreactivity to TNF-α was most pronounced in areas of liver injury and mostly restricted to large macrophages (F4/80 +) surrounded by a T-lymphocytic (CD3+) infiltrate. In vitro analysis of Kupffer cells to serial concentrations of LPS recapitulated the in vivo results, demonstrating up to 20-fold larger TNF-α levels in cells derived from sickle livers. To further elucidate the role of the liver macrophage in the in vivo immune response to LPS, sickle animals were challenged with LPS forty-eight hrs after Kupffer cell depletion with Gadolinium Chloride. Sickle mice treated with Gadolinium experienced enhanced survival and an ~90% reduction in serum TNF-α levels. In summary, the present study offers new insights into the responsiveness of the innate immune system in SCD to the highly conserved bacterial cell component, lipopolysaccharide. Unexpectedly, these data suggest that the liver macrophage in SCD, typically a cell type tolerant to the pro-inflammatory effects of LPS, has a cardinal role in orchestrating an excessive and harmful innate immune response to bacterial infections. Further studies will have to determine the immune response to other conserved bacterial structures and relate these findings to the human form of SCD.

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Prokaryotic ribosomal RNA stimulates zebrafish embryonic innate immune system

BMC Research Notes ◽

10.1186/s13104-019-4878-8 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Abhishikta Basu ◽

Maki Yoshihama ◽

Tamayo Uechi ◽

Naoya Kenmochi

Keyword(s):

Immune System ◽

Innate Immune System ◽

18S Rrna Gene ◽

Positive Control ◽

Innate Immune ◽

Rrna Gene ◽

Immune Recognition ◽

E Coli

Abstract Objectives Cell-culture studies reported that prokaryotic RNA molecules among the various microbe-associated molecular patterns (MAMPs) were uniquely present in live bacteria and were categorized as viability-associated MAMPs. They also reported that specific nucleotide modifications are instrumental in the discrimination between self and nonself RNAs. The aim of this study was to characterize the in vivo immune induction potential of prokaryotic and eukaryotic ribosomal RNAs (rRNAs) using zebrafish embryos as novel whole animal model system. Additionally, we aimed to test the possible role of rRNA modifications in immune recognition. Results We used three immune markers to evaluate the induction potential of prokaryotic rRNA derived from Escherichia coli and eukaryotic rRNAs from chicken (nonself) and zebrafish (self). Lipopolysaccharide (LPS) of Pseudomonas aeruginosa served as a positive control. E. coli rRNA had an induction potential equivalent to that of LPS. The zebrafish innate immune system could discriminate between self and nonself rRNAs. Between the nonself rRNAs, E. coli rRNA was more immunogenic than chicken rRNA. The in vitro transcript of zebrafish 18S rRNA gene without the nucleotide modifications was not recognized by its own immune system. Our data suggested that prokaryotic rRNA is immunostimulatory in vivo and could be useful as an adjuvant.

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Live Imaging of Disseminated Candidiasis in Zebrafish Reveals Role of Phagocyte Oxidase in Limiting Filamentous Growth

Eukaryotic Cell ◽

10.1128/ec.05005-11 ◽

2011 ◽

Vol 10 (7) ◽

pp. 932-944 ◽

Cited By ~ 80

Author(s):

Kimberly M. Brothers ◽

Zachary R. Newman ◽

Robert T. Wheeler

Keyword(s):

Immune System ◽

Nadph Oxidase ◽

Immune Cells ◽

Innate Immune System ◽

Innate Immune ◽

Yeast Cells ◽

Content Type ◽

Disseminated Candidiasis ◽

Phagocyte Nadph Oxidase

ABSTRACTCandida albicansis a human commensal and a clinically important fungal pathogen that grows in both yeast and hyphal forms during human infection. AlthoughCandidacan cause cutaneous and mucosal disease, systemic infections cause the greatest mortality in hospitals. Candidemia occurs primarily in immunocompromised patients, for whom the innate immune system plays a paramount role in immunity. We have developed a novel transparent vertebrate model of candidemia to probe the molecular nature ofCandida-innate immune system interactions in an intact host. Our zebrafish infection model results in a lethal disseminated disease that shares important traits with disseminated candidiasis in mammals, including dimorphic fungal growth, dependence on hyphal growth for virulence, and dependence on the phagocyte NADPH oxidase for immunity. Dual imaging of fluorescently marked immune cells and fungi revealed that phagocytosed yeast cells can remain viable and even divide within macrophages without germinating. Similarly, although we observed apparently killed yeast cells within neutrophils, most yeast cells within these innate immune cells were viable. Exploiting this model, we combined intravital imaging with gene knockdown to show for the first time that NADPH oxidase is required for regulation ofC. albicansfilamentationin vivo. The transparent and easily manipulated larval zebrafish model promises to provide a unique tool for dissecting the molecular basis of phagocyte NADPH oxidase-mediated limitation of filamentous growthin vivo.

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Bacillus anthracis Spores Stimulate Cytokine and Chemokine Innate Immune Responses in Human Alveolar Macrophages through Multiple Mitogen-Activated Protein Kinase Pathways

Infection and Immunity ◽

10.1128/iai.00446-06 ◽

2006 ◽

Vol 74 (8) ◽

pp. 4430-4438 ◽

Cited By ~ 28

Author(s):

Kaushik Chakrabarty ◽

Wenxin Wu ◽

J. Leland Booth ◽

Elizabeth S. Duggan ◽

K. Mark Coggeshall ◽

...

Keyword(s):

Immune System ◽

Bacillus Anthracis ◽

Alveolar Macrophages ◽

Innate Immune System ◽

Innate Immune ◽

Mitogen Activated Protein Kinase ◽

Tnf Α ◽

Human Alveolar Macrophages ◽

Mitogen Activated Protein ◽

Multiple Signaling

ABSTRACT Contact with the human alveolar macrophage plays a key role in the innate immune response to Bacillus anthracis spores. Because there is a significant delay between the initial contact of the spore with the host and clinical evidence of disease, there appears to be temporary containment of the pathogen by the innate immune system. Therefore, the early macrophage response to Bacillus anthracis exposure is important in understanding the pathogenesis of this disease. In this paper, we studied the initial events after exposure to spores, beginning with the rapid internalization of spores by the macrophages. Spore exposure rapidly activated the mitogen-activated protein kinase signaling pathways extracellular signal-regulated kinase, c-Jun-NH2-terminal kinase, and p38. This was followed by the transcriptional activation of cytokine and primarily monocyte chemokine genes as determined by RNase protection assays. Transcriptional induction is reflected at the translational level, as interleukin-1α (IL-1α), IL-1β, IL-6, and tumor necrosis factor alpha (TNF-α) cytokine protein levels were markedly elevated as determined by enzyme-linked immunosorbent assay. Induction of IL-6 and TNF-α, and, to a lesser extent, IL-1α and IL-1β, was partially inhibited by the blockade of individual mitogen-activated protein kinases, while the complete inhibition of cytokine induction was achieved when multiple signaling pathway inhibitors were used. Taken together, these data clearly show activation of the innate immune system in human alveolar macrophages by Bacillus anthracis spores. The data also show that multiple signaling pathways are involved in this cytokine response. This report is the first comprehensive examination of this process in primary human alveolar macrophages.

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The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.00925-16 ◽

2016 ◽

Vol 85 (3) ◽

Cited By ~ 2

Author(s):

Luis A. Vega ◽

Kayla M. Valdes ◽

Ganesh S. Sundar ◽

Ashton T. Belew ◽

Emrul Islam ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Immune Evasion ◽

Immune Cell ◽

Group A Streptococcus ◽

Transcriptional Regulator ◽

Innate Immune ◽

Content Type ◽

Human Host

ABSTRACTAs an exclusively human pathogen,Streptococcus pyogenes(the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene,cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators ofStreptococcus mutans(MetR),Streptococcus iniae(CpsY), andStreptococcus agalactiae(MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survivalin vivo. Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest thein vitrophenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE,speB,spd,nga[spn],prtS[SpyCEP], andsse) and cell surface-associated factors of GAS (emm1,mur1.2,sibA[cdhA], andM5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

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