PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (41)
 

Clipboard (0)
None

Select a Filter Below

Year of Publication
more »
1.  Complete Genome Sequence of Streptococcus agalactiae CNCTC 10/84, a Hypervirulent Sequence Type 26 Strain 
Genome Announcements  2014;2(6):e01338-14.
Streptococcus agalactiae (group B Streptococcus [GBS]) is a human pathogen with a propensity to cause neonatal infections. We report the complete genome sequence of GBS strain CNCTC 10/84, a hypervirulent clinical isolate frequently used to study GBS pathogenesis. Comparative analysis of this sequence may shed light on novel pathogenic mechanisms.
doi:10.1128/genomeA.01338-14
PMCID: PMC4276828  PMID: 25540350
2.  Human-Specific Bacterial Pore-Forming Toxins Induce Programmed Necrosis in Erythrocytes 
mBio  2014;5(5):e01251-14.
ABSTRACT
A subgroup of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins (PFTs) has an unusually narrow host range due to a requirement for binding to human CD59 (hCD59), a glycosylphosphatidylinositol (GPI)-linked complement regulatory molecule. hCD59-specific CDCs are produced by several organisms that inhabit human mucosal surfaces and can act as pathogens, including Gardnerella vaginalis and Streptococcus intermedius. The consequences and potential selective advantages of such PFT host limitation have remained unknown. Here, we demonstrate that, in addition to species restriction, PFT ligation of hCD59 triggers a previously unrecognized pathway for programmed necrosis in primary erythrocytes (red blood cells [RBCs]) from humans and transgenic mice expressing hCD59. Because they lack nuclei and mitochondria, RBCs have typically been thought to possess limited capacity to undergo programmed cell death. RBC programmed necrosis shares key molecular factors with nucleated cell necroptosis, including dependence on Fas/FasL signaling and RIP1 phosphorylation, necrosome assembly, and restriction by caspase-8. Death due to programmed necrosis in RBCs is executed by acid sphingomyelinase-dependent ceramide formation, NADPH oxidase- and iron-dependent reactive oxygen species formation, and glycolytic formation of advanced glycation end products. Bacterial PFTs that are hCD59 independent do not induce RBC programmed necrosis. RBC programmed necrosis is biochemically distinct from eryptosis, the only other known programmed cell death pathway in mature RBCs. Importantly, RBC programmed necrosis enhances the growth of PFT-producing pathogens during exposure to primary RBCs, consistent with a role for such signaling in microbial growth and pathogenesis.
IMPORTANCE
In this work, we provide the first description of a new form of programmed cell death in erythrocytes (RBCs) that occurs as a consequence of cellular attack by human-specific bacterial toxins. By defining a new RBC death pathway that shares important components with necroptosis, a programmed necrosis module that occurs in nucleated cells, these findings expand our understanding of RBC biology and RBC-pathogen interactions. In addition, our work provides a link between cholesterol-dependent cytolysin (CDC) host restriction and promotion of bacterial growth in the presence of RBCs, which may provide a selective advantage to human-associated bacterial strains that elaborate such toxins and a potential explanation for the narrowing of host range observed in this toxin family.
doi:10.1128/mBio.01251-14
PMCID: PMC4173772  PMID: 25161188
3.  α–Intercalated cells defend the urinary system from bacterial infection 
The Journal of Clinical Investigation  2014;124(7):2963-2976.
α–Intercalated cells (A-ICs) within the collecting duct of the kidney are critical for acid-base homeostasis. Here, we have shown that A-ICs also serve as both sentinels and effectors in the defense against urinary infections. In a murine urinary tract infection model, A-ICs bound uropathogenic E. coli and responded by acidifying the urine and secreting the bacteriostatic protein lipocalin 2 (LCN2; also known as NGAL). A-IC–dependent LCN2 secretion required TLR4, as mice expressing an LPS-insensitive form of TLR4 expressed reduced levels of LCN2. The presence of LCN2 in urine was both necessary and sufficient to control the urinary tract infection through iron sequestration, even in the harsh condition of urine acidification. In mice lacking A-ICs, both urinary LCN2 and urinary acidification were reduced, and consequently bacterial clearance was limited. Together these results indicate that A-ICs, which are known to regulate acid-base metabolism, are also critical for urinary defense against pathogenic bacteria. They respond to both cystitis and pyelonephritis by delivering bacteriostatic chemical agents to the lower urinary system.
doi:10.1172/JCI71630
PMCID: PMC4071397  PMID: 24937428
4.  Role of Pore-Forming Toxins in Bacterial Infectious Diseases 
SUMMARY
Pore-forming toxins (PFTs) are the most common bacterial cytotoxic proteins and are required for virulence in a large number of important pathogens, including Streptococcus pneumoniae, group A and B streptococci, Staphylococcus aureus, Escherichia coli, and Mycobacterium tuberculosis. PFTs generally disrupt host cell membranes, but they can have additional effects independent of pore formation. Substantial effort has been devoted to understanding the molecular mechanisms underlying the functions of certain model PFTs. Likewise, specific host pathways mediating survival and immune responses in the face of toxin-mediated cellular damage have been delineated. However, less is known about the overall functions of PFTs during infection in vivo. This review focuses on common themes in the area of PFT biology, with an emphasis on studies addressing the roles of PFTs in in vivo and ex vivo models of colonization or infection. Common functions of PFTs include disruption of epithelial barrier function and evasion of host immune responses, which contribute to bacterial growth and spreading. The widespread nature of PFTs make this group of toxins an attractive target for the development of new virulence-targeted therapies that may have broad activity against human pathogens.
doi:10.1128/MMBR.00052-12
PMCID: PMC3668673  PMID: 23699254
5.  Vaginolysin Drives Epithelial Ultrastructural Responses to Gardnerella vaginalis 
Infection and Immunity  2013;81(12):4544-4550.
Gardnerella vaginalis, the bacterial species most frequently isolated from women with bacterial vaginosis (BV), produces a cholesterol-dependent cytolysin (CDC), vaginolysin (VLY). At sublytic concentrations, CDCs may initiate complex signaling cascades crucial to target cell survival. Using live-cell imaging, we observed the rapid formation of large membrane blebs in human vaginal and cervical epithelial cells (VK2 and HeLa cells) exposed to recombinant VLY toxin and to cell-free supernatants from growing liquid cultures of G. vaginalis. Binding of VLY to its human-specific receptor (hCD59) is required for bleb formation, as antibody inhibition of either toxin or hCD59 abrogates this response, and transfection of nonhuman cells (CHO-K1) with hCD59 renders them susceptible to toxin-induced membrane blebbing. Disruption of the pore formation process (by exposure to pore-deficient toxoids or pretreatment of cells with methyl-β-cyclodextrin) or osmotic protection of target cells inhibits VLY-induced membrane blebbing. These results indicate that the formation of functional pores drives the observed ultrastructural rearrangements. Rapid bleb formation may represent a conserved response of epithelial cells to sublytic quantities of pore-forming toxins, and VLY-induced epithelial cell membrane blebbing in the vaginal mucosa may play a role in the pathogenesis of BV.
doi:10.1128/IAI.00627-13
PMCID: PMC3837968  PMID: 24082080
6.  DNase Inhibits Gardnerella vaginalis Biofilms In Vitro and In Vivo 
The Journal of Infectious Diseases  2013;207(10):1491-1497.
Bacterial vaginosis is a highly prevalent and poorly understood polymicrobial disorder of the vaginal microbiota, with significant adverse sequelae. Gardnerella vaginalis predominates in bacterial vaginosis. Biofilms of G. vaginalis are present in human infections and are implicated in persistent disease, treatment failure, and transmission. Here we demonstrate that G. vaginalis biofilms contain extracellular DNA, which is essential to their structural integrity. Enzymatic disruption of this DNA specifically inhibits biofilms, acting on both newly forming and established biofilms. DNase liberates bacteria from the biofilm to supernatant fractions and potentiates the activity of metronidazole, an antimicrobial agent used in the treatment of bacterial vaginosis. Using a new murine vaginal colonization model for G. vaginalis, we demonstrate >10-fold inhibition of G. vaginalis colonization by DNase. We conclude that DNase merits investigation as a potential nonantibiotic adjunct to existing bacterial vaginosis therapies in order to decrease the risk of chronic infection, recurrence, and associated morbidities.
doi:10.1093/infdis/jit047
PMCID: PMC3627197  PMID: 23431033
Bacterial vaginosis; biofilm; extracellular DNA; Gardnerella vaginalis
7.  Bordetella holmesii: initial genomic analysis of an emerging opportunist 
Pathogens and disease  2013;67(2):132-135.
Bordetella holmesii is an emerging opportunistic pathogen that causes respiratory disease in healthy individuals and invasive infections among patients lacking splenic function. We used 16S rRNA analysis to confirm B. holmesii as the cause of bacteremia in a child with sickle cell disease. Semiconductor-based draft genome sequencing provided insight into B. holmesii phylogeny and potential virulence mechanisms and also identified a toluene-4-monoxygenase locus unique among bordetellae.
doi:10.1111/2049-632X.12028
PMCID: PMC3653170  PMID: 23620158
Bordetella holmesii; genome; asplenia; opportunistic
8.  Case Report: Group B Streptococcus meningitis in an adolescent    
F1000Research  2014;3:167.
Streptococcus agalactiae (group B Streptococcus, GBS) usually colonizes the gastrointestinal and lower genital tracts of asymptomatic hosts, yet the incidence of invasive disease is on the rise . We describe a case of an 18 year old woman, recently diagnosed with lupus, who reported a spontaneous abortion six weeks prior to her hospitalization.  She presented with fever, altered mental status, and meningeal signs, paired with a positive blood culture for GBS. Magnetic resonance imaging of her brain demonstrated an extra-axial fluid collection, and she was diagnosed with meningitis.  She received prolonged intravenous antibiotic therapy and aggressive treatment for lupus, leading to clinical recovery. This case illustrates the importance of recognizing GBS as a potential pathogen in all patients presenting with CNS infection .  
doi:10.12688/f1000research.4651.1
PMCID: PMC4193390  PMID: 25339988
9.  Klebsiella pneumoniae K1 Liver Abscess and Septic Endophthalmitis in a U.S. Resident 
Journal of Clinical Microbiology  2013;51(3):1049-1051.
Klebsiella pneumoniae K1 is a major agent of hepatic abscess with metastatic disease in East Asia, with sporadic reports originating elsewhere. We report a case of abscess complicated by septic endophthalmitis caused by a wzyAKpK1-positive Klebsiella strain in a U.S. resident, raising concern for global emergence.
doi:10.1128/JCM.02853-12
PMCID: PMC3592058  PMID: 23303492
10.  Cigarette Smoke Increases Staphylococcus aureus Biofilm Formation via Oxidative Stress 
Infection and Immunity  2012;80(11):3804-3811.
The strong epidemiological association between cigarette smoke (CS) exposure and respiratory tract infections is conventionally attributed to immunosuppressive and irritant effects of CS on human cells. Since pathogenic bacteria such as Staphylococcus aureus are members of the normal microbiota and reside in close proximity to human nasopharyngeal cells, we hypothesized that bioactive components of CS might affect these organisms and potentiate their virulence. Using Staphylococcus aureus as a model organism, we observed that the presence of CS increased both biofilm formation and host cell adherence. Analysis of putative molecular pathways revealed that CS exposure decreased expression of the quorum-sensing agr system, which is involved in biofilm dispersal, and increased transcription of biofilm inducers such as sarA and rbf. CS contains bioactive compounds, including free radicals and reactive oxygen species, and we observed transcriptional induction of bacterial oxidoreductases, including superoxide dismutase, following exposure. Moreover, pretreatment of CS with an antioxidant abrogated CS-mediated enhancement of biofilms. Exposure of bacteria to hydrogen peroxide alone increased biofilm formation. These observations are consistent with the hypothesis that CS induces staphylococcal biofilm formation in an oxidant-dependent manner. CS treatment induced transcription of fnbA (encoding fibronectin binding protein A), leading to increased binding of CS-treated staphylococci to immobilized fibronectin and increased adherence to human cells. These observations indicate that the bioactive effects of CS may extend to the resident microbiota of the nasopharynx, with implications for the pathogenesis of respiratory infection in CS-exposed humans.
doi:10.1128/IAI.00689-12
PMCID: PMC3486061  PMID: 22890993
11.  Lactobacillus crispatus Dominant Vaginal Microbiome Is Associated with Inhibitory Activity of Female Genital Tract Secretions against Escherichia coli 
PLoS ONE  2014;9(5):e96659.
Objective
Female genital tract secretions inhibit E. coli ex vivo and the activity may prevent colonization and provide a biomarker of a healthy microbiome. We hypothesized that high E. coli inhibitory activity would be associated with a Lactobacillus crispatus and/or jensenii dominant microbiome and differ from that of women with low inhibitory activity.
Study Design
Vaginal swab cell pellets from 20 samples previously obtained in a cross-sectional study of near-term pregnant and non-pregnant healthy women were selected based on having high (>90% inhibition) or low (<20% inhibition) anti-E. coli activity. The V6 region of the 16S ribosomal RNA gene was amplified and sequenced using the Illumina HiSeq 2000 platform. Filtered culture supernatants from Lactobacillus crispatus, Lactobacillus iners, and Gardnerella vaginalis were also assayed for E. coli inhibitory activity.
Results
Sixteen samples (10 with high and 6 with low activity) yielded evaluable microbiome data. There was no difference in the predominant microbiome species in pregnant compared to non-pregnant women (n = 8 each). However, there were significant differences between women with high compared to low E. coli inhibitory activity. High activity was associated with a predominance of L. crispatus (p<0.007) and culture supernatants from L. crispatus exhibited greater E. coli inhibitory activity compared to supernatants obtained from L. iners or G. vaginalis. Notably, the E. coli inhibitory activity varied among different strains of L. crispatus.
Conclusion
Microbiome communities with abundant L. crispatus likely contribute to the E. coli inhibitory activity of vaginal secretions and efforts to promote this environment may prevent E. coli colonization and related sequelae including preterm birth.
doi:10.1371/journal.pone.0096659
PMCID: PMC4013016  PMID: 24805362
12.  Genome Sequence of the Human Abscess Isolate Streptococcus intermedius BA1 
Genome Announcements  2013;1(1):e00117-12.
Streptococcus intermedius is a human pathogen with a propensity for abscess formation. We report a high-quality draft genome sequence of S. intermedius strain BA1, an isolate from a human epidural abscess. This sequence provides insight into the biology of S. intermedius and will aid investigations of pathogenicity.
doi:10.1128/genomeA.00117-12
PMCID: PMC3569275  PMID: 23405291
13.  Genome Sequence of Bacterial Interference Strain Staphylococcus aureus 502A 
Genome Announcements  2014;2(2):e00284-14.
Staphylococcus aureus 502A was a strain used in bacterial interference programs during the 1960s and early 1970s. Infants were deliberately colonized with 502A with the goal of preventing colonization with more invasive strains. We present the completed genome sequence of this organism.
doi:10.1128/genomeA.00284-14
PMCID: PMC3983310  PMID: 24723721
14.  Emergence of the Epidemic Methicillin-Resistant Staphylococcus aureus Strain USA300 Coincides with Horizontal Transfer of the Arginine Catabolic Mobile Element and speG-mediated Adaptations for Survival on Skin 
mBio  2013;4(6):e00889-13.
ABSTRACT
The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone.
IMPORTANCE
Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells.
doi:10.1128/mBio.00889-13
PMCID: PMC3870260  PMID: 24345744
15.  Retrocyclin inhibits Gardnerella vaginalis biofilm formation and toxin activity 
Journal of Antimicrobial Chemotherapy  2012;67(12):2870-2872.
Background
Retrocyclins are cyclic antimicrobial peptides that have been shown to be both broadly active and safe in animal models. RC-101, a synthetic retrocyclin, targets important human pathogens and is a candidate vaginal microbicide. Its activity against microbes associated with bacterial vaginosis is unknown.
Methods
We investigated the effect of RC-101 on toxin activity, bacterial growth and biofilm formation of Gardnerella vaginalis in vitro.
Results
RC-101 potently inhibits the cytolytic activity of vaginolysin, the Gardnerella vaginalis toxin, on both erythrocytes and nucleated cells. RC-101 lacks inhibitory activity against planktonic G. vaginalis but markedly decreases biofilm formation.
Conclusions
These dual properties, toxin inhibition and biofilm retardation, justify further exploration of RC-101 as a candidate agent for bacterial vaginosis prevention.
doi:10.1093/jac/dks305
PMCID: PMC3494843  PMID: 22855857
defensin; vaginolysin; bacterial vaginosis; biofilm
16.  Crystal structures of respiratory pathogen neuraminidases 
Biochemical and biophysical research communications  2009;380(3):10.1016/j.bbrc.2009.01.108.
Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7 Å resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.
doi:10.1016/j.bbrc.2009.01.108
PMCID: PMC3836282  PMID: 19284989
Streptococcus pneumoniae; Pseudomonas aeruginosa; neuraminidase; crystal structure; pneumonia
17.  β-Hemolysin/Cytolysin of Group B Streptococcus Enhances Host Inflammation but Is Dispensable for Establishment of Urinary Tract Infection 
PLoS ONE  2013;8(3):e59091.
Group B Streptococcus (GBS; Streptococcus agalactiae) is a major human pathogen that disproportionately affects neonates and women in the peripartum period and is an emerging cause of infection in older adults. The primary toxin of GBS, β-hemolysin/cytolysin (βH/C), has a well-defined role in the pathogenesis of invasive disease, but its role in urinary tract infection (UTI) is unknown. Using both in vitro and in vivo models, we analyzed the importance of βH/C in GBS uropathogenesis. There were no significant differences in bacterial density from the bladders or kidneys from mice infected with wild-type or isogenic βH/C-deficient GBS, and competitive indices from co-infection experiments were near 1. Thus, βH/C is dispensable for the establishment of GBS-UTI. However, βH/C-sufficient GBS induced a more robust proinflammatory cytokine response in cultured bladder epithelial cells and in the urinary tracts of infected mice. Given the near ubiquity of βH/C-expressing strains in epidemiologic studies and the importance of local inflammation in dictating outcomes and sequelae of UTI, we hypothesize that βH/C-driven inflammatory signaling may be important in the clinical course of GBS-UTI.
doi:10.1371/journal.pone.0059091
PMCID: PMC3591438  PMID: 23505569
18.  Predictors of Staphylococcus aureus Rectovaginal Colonization in Pregnant Women and Risk for Maternal and Neonatal Infections 
Background.
Staphylococcus aureus infections are increasing among pregnant and postpartum women and neonates, but risk factors for S. aureus colonization in pregnancy and the association between maternal colonization and infant infections are not well defined. We sought to identify risk factors for maternal S. aureus rectovaginal colonization and assess colonization as a risk factor for infections among mothers and infants.
Methods.
We conducted a retrospective cohort study of pregnant women and their infants. Demographic and clinical data, including S. aureus infections that occurred in mothers from 3 months before to 3 months after delivery and in infants during the first 3 months of life, were extracted from electronic medical records. Predictors for maternal S. aureus rectovaginal colonization were assessed through multivariable logistic regression analysis.
Results.
The cohort included 2702 women and 2789 infants. The prevalence of maternal rectovaginal colonization with methicillin-susceptible S. aureus and methicillin-resistant S. aureus (MRSA) was 13% and 0.7%. Independent predictors of colonization included multigravidity, human immunodeficiency virus seropositivity, and group B Streptococcus colonization. S. aureus colonization was associated with an increased risk of infection in mothers (odds ratio [OR], 3.5; 95% confidence interval [CI], 1.4–8.8) but not in their infants (OR, 1.9; 95% CI, .6–5.6). The frequency of S. aureus infections was 0.8% in mothers and 0.7% in infants.
Conclusions.
S. aureus rectovaginal colonization was associated with an increased risk of infections in women but not in their infants. The frequency of MRSA infections was low. These data suggest that routine MRSA screening of pregnant women may not be indicated.
doi:10.1093/jpids/pis001
PMCID: PMC3656550  PMID: 23687569
19.  Microbiota of the upper and lower genital tract 
Summary
Our understanding of the bacterial species inhabiting the female genital tract has been limited primarily by our ability to detect them. Early investigations using microscopy and culture-based techniques identified lactobacilli as the predominant members of the vaginal microbiota and suggested that these organisms might serve a protective function at the mucosal surface. Improvements in cultivation techniques and the development of molecular-based detection strategies validated these early findings and enabled us to recognize that the microbiota of the female genital tract is much more complex than previously suspected. Disruption of the vaginal microbial community due to invasion of exogenous organisms or by overgrowth of one or more endogenous species has important health implications for both the mother and newborn.
doi:10.1016/j.siny.2011.08.006
PMCID: PMC3242913  PMID: 21920833
Bacterial vaginosis; Lactobacilli; Vaginal microbiota; Vaginitis
20.  Nod1 mediates cytoplasmic sensing of combinations of extracellular bacteria 
Cellular microbiology  2007;9(5):1343-1351.
During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defense. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signaling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore-forming toxin pneumolysin from S. pneumoniae. Radiolabeled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore-forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureus α-toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non-pore-forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan-recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.
doi:10.1111/j.1462-5822.2006.00878.x
PMCID: PMC1867455  PMID: 17474907
21.  EPITHELIAL CELLS ARE SENSITIVE DETECTORS OF BACTERIAL PORE-FORMING TOXINS 
The Journal of biological chemistry  2006;281(18):12994-12998.
Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins – including pneumolysin from Streptococcus pneumoniae, α-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis – at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial PFT, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.
doi:10.1074/jbc.M511431200
PMCID: PMC1586115  PMID: 16520379
22.  Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum 
BMC Microbiology  2011;11:239.
Background
Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis, wound infections, and a variety of occasional invasive diseases. Since its initial discovery in 1946, this Gram positive organism has been known to have hemolytic activity, yet no hemolysin has been previously reported. A. haemolyticum also displays variable hemolytic activity on laboratory blood agar that is dependent upon which species the blood is derived.
Results
Here we describe a cholesterol-dependent cytolysin (CDC) secreted by A. haemolyticum, designated arcanolysin (aln), which is present in all strains (n = 52) tested by DNA dot hybridization. Among the known CDCs, ALN is most closely related to pyolysin (PLO) from Trueperella (formerly Arcanobacterium) pyogenes. The aln probe, however, did not hybridize to DNA from T. pyogenes. The aln open reading frame has a lower mol %G+C (46.7%) than the rest of the A. haemolyticum genome (53.1%) and is flanked by two tRNA genes, consistent with probable acquisition by horizontal transfer. The ALN protein (~ 64 kDa) contains a predicted signal sequence, a putative PEST sequence, and a variant undecapeptide within domain 4, which is typically important for function of the toxins. The gene encoding ALN was cloned and expressed in Escherichia coli as a functional recombinant toxin. Recombinant ALN had hemolytic activity on erythrocytes and cytolytic activity on cultured cells from human, rabbit, pig and horse origins but was poorly active on ovine, bovine, murine, and canine cells. ALN was less sensitive to inhibition by free cholesterol than perfringolysin O, consistent with the presence of the variant undecapeptide.
Conclusions
ALN is a newly identified CDC with hemolytic activity and unique properties in the CDC family and may be a virulence determinant for A. haemolyticum.
doi:10.1186/1471-2180-11-239
PMCID: PMC3215231  PMID: 22029628
23.  Inerolysin, a Cholesterol-Dependent Cytolysin Produced by Lactobacillus iners▿  
Journal of Bacteriology  2010;193(5):1034-1041.
Lactobacillus iners is a common constituent of the human vaginal microbiota. This species was only recently characterized due to its fastidious growth requirements and has been hypothesized to play a role in the pathogenesis of bacterial vaginosis. Here we present the identification and molecular characterization of a protein toxin produced by L. iners. The L. iners genome encodes an open reading frame with significant primary sequence similarity to intermedilysin (ILY; 69.2% similarity) and vaginolysin (VLY; 68.4% similarity), the cholesterol-dependent cytolysins from Streptococcus intermedius and Gardnerella vaginalis, respectively. Clinical isolates of L. iners produce this protein, inerolysin (INY), during growth in vitro, as assessed by Western analysis. INY is a pore-forming toxin that is activated by reducing agents and inhibited by excess cholesterol. It is active across a pH range of 4.5 to 6.0 but is inactive at pH 7.4. At sublytic concentrations, INY activates p38 mitogen-activated protein kinase and allows entry of fluorescent phalloidin into the cytoplasm of epithelial cells. Unlike VLY and ILY, which are human specific, INY is active against cells from a broad range of species. INY represents a new target for studies directed at understanding the role of L. iners in states of health and disease at the vaginal mucosal surface.
doi:10.1128/JB.00694-10
PMCID: PMC3067590  PMID: 21169489
24.  The Ngal Reporter Mouse Detects the Response of the Kidney to Injury in Real Time 
Nature medicine  2011;17(2):216-222.
Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential characteristics which link the protein to the injured organ have not yet been described. We generated an NGAL-reporter mouse by inserting a di-fusion reporter gene, Luciferase2(Luc2)/mCherry(mC) into the Ngal locus. The Ngal-Luc2/mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real-time. In the kidney, Ngal-Luc2/mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ and cellular specific relationship with tubular stress, which quantitatively paralleled urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2/mC mice could also track both the onset and the resolution of the injury, and monitor the actions of NF-κB inhibitors and antibiotics in the case of infection. Accordingly, the imaging of Ngal-Luc2/mC mice and cells identified injurious and reparative agents which effect kidney damage.
doi:10.1038/nm.2290
PMCID: PMC3059503  PMID: 21240264
25.  Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract 
mBio  2011;2(3):e00016-11.
ABSTRACT
The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4−/−, Myd88−/−, Trif−/−, and Nod2−/− mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors.
IMPORTANCE
The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.
doi:10.1128/mBio.00016-11
PMCID: PMC3101776  PMID: 21586648

Results 1-25 (41)