Infection by the human fungal pathogen Aspergillus fumigatus induces hypoxic microenvironments within the lung that can alter the course of fungal pathogenesis. How hypoxic microenvironments shape the composition and immune activating potential of the fungal cell wall remains undefined. Herein we demonstrate that hypoxic conditions increase the hyphal cell wall thickness and alter its composition particularly by augmenting total and surface-exposed β-glucan content. In addition, hypoxia-induced cell wall alterations increase macrophage and neutrophil responsiveness and antifungal activity as judged by inflammatory cytokine production and ability to induce hyphal damage. We observe that these effects are largely dependent on the mammalian β-glucan receptor dectin-1. In a corticosteroid model of invasive pulmonary aspergillosis, A. fumigatus β-glucan exposure correlates with the presence of hypoxia in situ. Our data suggest that hypoxia-induced fungal cell wall changes influence the activation of innate effector cells at sites of hyphal tissue invasion, which has potential implications for therapeutic outcomes of invasive pulmonary aspergillosis.
Aspergillus fumigatus; hypoxia; beta-glucan; fungal pathogenesis; cell wall
One of the prototype mammalian kinases is PKA and various roles have been defined for PKA in malaria pathogenesis. The recently described phospho-proteomes of Plasmodium falciparum introduced a great volume of phospho-peptide data for both basic research and identification of new anti-malaria therapeutic targets. We discuss the importance of phosphorylations detected in vivo at different sites in the parasite R and C subunits of PKA and highlight the inhibitor sites in the parasite R subunit. The N-terminus of the parasite R subunit is predicted to be very flexible and we propose that phosphorylation at multiple sites in this region likely represent docking sites for interactions with other proteins, such as 14-3-3. The most significant observation when the P. falciparum C subunit is compared to mammalian C isoforms is lack of phosphorylation at a key site tail implying that parasite kinase activity is not regulated so tightly as mammalian PKA. Phosphorylation at sites in the activation loop could be mediating a number of processes from regulating parasite kinase activity, to mediating docking of other proteins. The important differences between Plasmodium and mammalian PKA isoforms that indicate the parasite kinase is a valid anti-malaria therapeutic target.
Plasmodium; Malaria; Kinase; cAMP-dependent protein kinase; Phosphorylation; Therapeutic target
Chlamydia trachomatis, although commonly asymptomatic in women, can result in chronic sequelae, such as pelvic inflammatory disease, ectopic pregnancy and infertility. However, a clear relationship has not been determined between specific serovars and the ability to lead to upper genital tract infection or infertility. Thus, in order to investigate differences in pathogenicity, C3H/HeN mice were infected in the ovarian bursa with the C. trachomatis strains D (UW-3/Cx), F (N.I.1), F (IC-Cal-3) and E (Bour). Differences both in the amount of vaginal shedding as well as subsequent fertility rates were observed between D (UW-3/Cx) and F (N.I.1) compared to F (IC-Cal-3) and E (Bour). Approximately 50% of the mice infected with the D (UW-3/Cx) and F (N.I.1) strains had vaginal shedding for up to 3–4 weeks after infection and fertility rates of less than 25%. Furthermore, mice inoculated with D (UW-3/Cx) and F (N.I.1) showed infertility even in the absence of medroxy progesterone acetate (MPA) treatment. In contrast, both MPA and non-MPA treated mice infected with F (IC-Cal-3) or E (Bour) did not show vaginal shedding and had fertility rates between 45–88%. Mutations in the CT135 open reading frame have been associated with virulence. However, no nucleotide differences were found among the four isolates for CT135. This murine model of infection with C. trachomatis may help with the understanding of disease pathology in humans and ultimately vaccine development.
Chlamydia; infertility; murine; pathogenicity
The blood feeding behavior of disease-transmitting arthropods creates a unique intersection between vertebrate and invertebrate physiology. Here, we review host blood-derived factors that persist through blood digestion to affect the lifespan, reproduction, and immune responses of some of the most common arthropod vectors of human disease.
tick; mosquito; Plasmodium; insulin; transforming growth factor-beta (TGF-β); macrophage migration inhibitory factor (MIF)
Professional phagocytes digest internalized microorganisms by actively delivering them into the phagolysosomal compartment. Intravacuolar bacterial pathogens have evolved a variety of effective strategies to bypass the default pathway of phagosomal maturation to create a niche permissive for their survival and propagation. Here we discuss recent progress in our understanding of the sophisticated mechanisms used by Legionella pneumophila to survive in phagocytes.
intracellular pathogens; vesicle trafficking; type IV secretion; effectors; host function subversion
Both anti-viral and anti-bacterial host defense mechanisms involve TRIF signaling. TRIF provides early clearance of pathogens and coordination of a local inflammatory ensemble through an interferon cascade, while it may trigger organ damage. The multipotentiality of TRIF-mediated immune machinery may direct the fate of our continuous battle with microbes.
Host defense mechanism; innate immunity; toll-like receptor; TRIF
Pseudomonas aeruginosa, a significant cause of human morbidity and mortality, uses a type 3 secretion system (T3SS) to inject effector toxins into host cells. We previously reported that P. aeruginosa uses ADP-ribosyltransferase (ADPr) activity of the T3SS effector ExoS for intracellular replication. T3SS translocon (ΔpopB)-mutants, which can export, but not translocate effectors across host membranes, retained intracellular replication. We hypothesized that secreted effectors mediate translocon-independent intracellular replication. Translocon mutants of PAO1 lacking one or more of its three known effectors (ExoS, ExoT and ExoY) were used. All translocon mutants, irrespective of effectors expressed, localized to intracellular vacuoles. Translocon-effector null mutants and translocon-exoS mutants showed defective intracellular replication. Mutants in exoT, exoY or both replicated as efficiently as translocon mutants expressing all effectors. Complementation of translocon-effector null mutants with native exoS or a membrane localization domain mutant of exoS, but not the ADPr mutant exoS (pUCPexoSE381D), restored intracellular replication, correlating with increased bacteria per vacuole. Thus, P. aeruginosa is capable of intravacuolar replication that requires ExoS ADPr activity, but not the translocon. These data suggest that T3SS effectors can participate in pathogenesis without translocon-mediated translocation across host membranes, and that intracellular bacteria can contribute to P. aeruginosa pathogenesis within epithelial cells.
Pseudomonas aeruginosa; intracellular replication; ExoS; ADPr domain; translocon; vacuole; epithelial cells
Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications.
In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A2B receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O2 release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A2A receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD.
Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and progression of disease. Thus, adenosine signaling represents a potentially important therapeutic target for the treatment and prevention of disease.
sickle cell disease; malaria; adenosine; adenosine A2B receptor; 2,3-diphosphoglycerate; adenosine deaminase
H. pylori infection is highly prevalent in Chile (73%). Usually a minority of infected patients develops complications such as ulcers and gastric cancer that have been associated with the presence of virulence factors (cagA, vacA) and host T helper response (Th1/Th2). Our aim was to evaluate the relationship between strain virulence and host immune response, using a multiple regression approach for the development of a model based on data collected from H. pylori infected patients in Chile. We analyzed levels of selected cytokines determined by ELISA (IL-12, IL-10, IFN-γ and IL-4) and the presence of cagA and vacA alleles polymorphisms determined by PCR in antral biopsies of 41 patients referred to endoscopy. By multiple regression analysis we established a correlation between bacterial and host factors using clinical outcome (gastritis and duodenal ulcer) as dependent variables. The selected model was described by: clinical outcome = 0.867491 (cagA) + 0.0131847 (IL-12/IL-10) + 0.0103503 (IFN-γ/IL-4) and it was able to explain over 90% of clinical outcomes observations (R2=96.4). This model considers that clinical outcomes are better explained by the interaction of host immune factors and strain virulence as a complex and interdependent mechanism.
H. pylori; virulence factors; cytokines; gastroduodenal ulcer
Chlamydia, like other intracellular bacteria, are auxotrophic for a variety of essential metabolites and obtain cholesterol and fatty acids from their eukaryotic host cell, however not many Chlamydia-specific enzymes have been identified that are involved in lipid metabolism. In silico analysis of one candidate C. trachomatis enzyme, annotated as a conserved putative hydrolase (CT149), identified two lipase/esterase GXSXG motifs, and a potential cholesterol recognition/interaction amino acid consensus (CRAC) sequence. His-tag purified recombinant CT149 exhibited ester hydrolysis activity in a nitrophenyl acetate-based cell-free assay system. When cholesteryl linoleate was used as substrate, ester hydrolysis occurred and production of cholesterol was detected by high performance liquid chromatography. Exogenous expression of transfected CT149 in HeLa cells resulted in a significant decrease of cytoplasmic cholesteryl esters within 48 hrs. These results demonstrate that CT149 has cholesterol esterase activity and is likely to contribute to the hydrolysis of eukaryotic cholesteryl esters during intracellular chlamydial growth.
Chlamydia; lipids; cholesterol esterase; cholesteryl esters
The early host response during pulmonary nocardiosis is highly dependent on neutrophils and the successful clearance of bacteria in tissue. The data presented in this study showed that IL-17 mediated the neutrophil response following intranasal inoculation with Nocardia asteroides strain GUH-2. Flow cytometry revealed that neutrophil levels in C57BL/6 mice were increased by day 1 post inoculation and remained elevated until day 3, during which time the majority of bacterial clearance occurred. Intracellular cytokine staining for IL-17 showed a 3.5- to 5-fold increase in IL-17 producing T-lymphocytes that were predominately comprised by CD4−CD8− γδ T-lymphocytes. The importance of IL-17 and γδ T-cells was determined by the in vivo administration of antibody, capable of blocking IL-17 binding or TCR δ, respectively. Neutralization of either IL-17 or γδ T-cells in Nocardia treated mice resulted in attenuated neutrophil infiltration. Paralleling this impaired neutrophil recruitment, nearly a 10-fold increase in bacterial burden was observed in both anti-IL-17 and anti-TCR δ treated animals. Together, these data indicate a protective role for IL-17 and suggest that IL-17 producing γδ T-lymphocytes contribute to neutrophil infiltration during pulmonary nocardiosis.
INTERLEUKIN-17; NEUTROPHIL; T-LYMPHOCYTES; NOCARDIOSIS
Chlamydiae are important human pathogens that are responsible for a wide rage of diseases with a significant impact on public health. In this review article we highlight how recent studies have increased our knowledge of Chlamydia pneumoniae pathogenesis and mechanisms of innate immunity directed host defense against Chlamydia pneumoniae infection.
Chlamydia pneumoniae; TLR; NLR; inflammasome
The importance of innate immunity lies not only in directly confronting pathogenic and non-pathogenic insults but also in instructing the development of an efficient adaptive immune response. The Nlrp3 inflammasome provides a platform for the activation of caspase-1 with the subsequent processing and secretion of IL-1 family members. Given the importance of IL-1 in a variety of inflammatory diseases, understanding the role of Nlrp3 inflammasome in the initiation of innate and adaptive immune responses cannot be overstated. This review examines recent advances in inflammasome biology with an emphasis on its roles in sterile inflammation and triggering of adaptive immune responses.
inflammasome; innate immunity; adaptive immunity
There is considerable evidence that phase variation among transparent and opaque colony phenotypes of Streptococcus pneumoniae (Spn) plays an important role in the pneumococcal adherence and invasion. The current study was designed to investigate the interactions of the opacity phenotype variants of Spn with specific complement pathway activation in a mouse model of acute otitis media (AOM). Although the opaque colony phenotype was expected to be more resistant to complement mediated killing compared to the transparent Spn variant, we discovered that C3b deposition on the transparent Spn is, in large part, dependent on the alternative pathway activation. There were no significant differences in resistance to complement mediated opsonophagocytosis between the two variants in factor B deficient mice. In addition, an in vitro study demonstrated that significantly more C4b-binding protein (C4BP) (the classical pathway inhibitor) and factor H (FH) (the alternative pathway inhibitor) bound to the transparent strain compared with the opaque one. Our data suggest that the difference in the relative virulence of Spn opacity phenotypes is associated with its ability to evade complement-mediated opsonophagocytosis in a mouse model of pneumococcal AOM.
Streptococcus pneumoniae; colony opacity variants; complement; otitis media
In culture, exposure to penicillin and other stressors induce chlamydiae to enter a non-infectious but viable state termed persistence. Chlamydiae may reenter their normal developmental cycle after stressor removal. Though aberrant RB similar to those present in culture models of persistence have been observed within infected tissues, the existence of persistent chlamydiae has not been definitively demonstrated in vivo. As a result, the role of persistent organisms in pathogenesis is undefined. In order to establish an experimentally tractable model of in vivo persistence, C. muridarum vaginally-infected mice were gavaged with either water or amoxicillin (amox). Vaginal swabs were collected for chlamydial titration and RNA isolated for quantification of pre-16s rRNA. Uterine tissue was analyzed by transmission electron microscopy (TEM). Although amox-treatment reduced vaginal shedding by >99%, C. muridarum pre-16s rRNA accumulation was unchanged by treatment. These data indicate that the amox-exposed organisms were viable but not infectious. Furthermore, TEM analyses demonstrated that inclusions in amox-treated animals contained primarily large, aberrant RB, but those observed in untreated control animals were normal. Collectively, these data suggest that amoxicillin treatment induces C. muridarum to enter the persistent state in vivo. This model also represents the first experimentally tractable animal model of chlamydial persistence.
C. trachomatis; C. muridarum; chlamydial persistence; aberrant reticulate body; aberrant body; amoxicillin
Long-term protection against Toxoplasma gondii is dependent on robust CD8+ T cell immunity. In the absence of this response, the host is unable to maintain chronicity, which results in recrudescence of infection and possible death. Factors needed for the persistence of protective CD8+ T cells against the parasite need to be evaluated. Previous studies from our laboratory have reported that synergism between γ chain cytokines like IL-7 and IL-15 is critical for the generation of CD8+ T cell response needed for protection during acute infection. In this study we report that the situation is different during the recall response where CD8+ T cell response is almost entirely dependent on IL-15, with IL-7 at best playing a minor role. In the absence of IL-15, CD8+ T cells fail to respond optimally to parasitic re-challenge and hosts are unable to control their replication, which leads to their death. Thus T. gondii infection may represent a unique situation where CD8+ T cell response during secondary challenge is primarily dependent on IL-15 with other γ chain cytokines having nominal effect. These findings provide important information regarding factors involved in the generation of protective immunity against T.gondii with strong implications in developing immunotherapeutic agents against the pathogen.
Toxoplasma gondii; CD8+ T cells; IL-15; IL-7
Humoral immune mechanisms are an important component of protective immunity to Ehrlichia species. However, the molecular basis of antibody mediated immunity is not completely defined, and the role of other molecularly characterized major immunoreactive proteins is unknown. In previous studies, we mapped major species-specific continuous epitopes in three surface exposed and secreted tandem repeat proteins (TRP32, TRP47 and TRP120). In this study, we report that protection is provided by antibodies against these molecularly defined TRP epitopes using in vitro and in vivo models. Protection was demonstrated in vitro after prophylactic and therapeutic administration of epitope-specific anti-TRP antibodies, suggesting that the protective mechanisms involve extracellular and intracellular antibody-mediated effects. In vivo passive transfer of individual epitope-specific TRP sera significantly reduced the ehrlichial load and splenomegaly, and protected mice against lethal infection. Moreover, the combination of antibodies to all three TRPs provided enhanced reduction in ehrlichial load similar to that of E. chaffeensis immune sera. IgG1 was the predominant antibody isotype in the epitope-specific TRP mouse sera. These results demonstrate that antibodies against linear epitopes in TRP32, TRP47 and TRP120 are protective during E. chaffeensis infection and involves extracellular and intracellular antibody-mediated mechanisms.
EHRLICHIA CHAFFEENSIS; TANDEM REPEAT PROTEINS; IMMUNOREACTIVE PROTEINS; EPITOPE; ANTIBODY
Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in pathogenesis and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was prepared on a wild type pneumococcal TIGR4 background. When the mutant and wild type strains were compared for adherence to D562 and A549 cell lines a reduction in adherence by the mutant was observed (p= 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host E coli resulting in augmented adherence to D562 (p= 0.002) and A549 (p= 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p=0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p=0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.
Rabbits, mice, rats, non-human primates, sheep and cattle have been used to study the effect of Clostridium perfringens enterotoxin (CPE). CPE produces mostly necrosis of the small intestinal epithelium along with fluid accumulation in rabbits and mice. In the latter, CPE can bind to internal organs such as the liver, which induces lethal potassium levels in blood.
animal models; calf; Clostridium perfringens; enterotoxin; lamb; mouse; non-human primate; rabbit; rat
The use of iron as an enzymatic cofactor is pervasive in biological systems. Consequently most living organisms, including pathogenic bacteria, require iron to survive and replicate. To combat infection vertebrates have evolved sophisticated iron sequestration systems against which, pathogenic bacteria have concomitantly evolved equally elaborate iron acquisition mechanisms.
Bacterial pathogenesis; Staphylococcus aureus; Isd system; nutritional immunity; Staphylococcal iron acquisition
Staphylococcus aureus is a highly virulent bacterial pathogen capable of causing a variety of ailments throughout the human body. It is a major public health concern due to the continued emergence of highly pathogenic methicillin resistant strains (MRSA) both within hospitals and in the community. Virulence in S. aureus is mediated by an array of secreted and cell wall associated virulence factors, including toxins, hemolysins and proteases. In this work we identify a leucine aminopeptidase (LAP, pepZ) that strongly impacts the pathogenic abilities of S. aureus. Disruption of the pepZ gene in either Newman or USA300 resulted in a dramatic attenuation of virulence in both localized and systemic models of infection. LAP is required for survival inside human macrophages and gene expression analysis shows that pepZ expression is highest in the intracellular environment. We examine the cellular location of LAP and demonstrate that it is localized to the bacterial cytosol. These results identify for the first time an intracellular leucine aminopeptidase that influences disease causation in a Gram-positive bacterium.
Leucine aminopeptidase; LAP; virulence; peptidase; staphylococcus
Human immunodeficiency virus type 1 (HIV-1) mostly owes its success to its ability to evade host immune responses. Understanding viral immune escape mechanisms is prerequisite to improve future HIV-1 vaccine design. This review focuses on the strategies that HIV-1 has evolved to evade recognition by natural killer (NK) cells.
HIV-1; NK cells; escape; innate immunity; vaccine
The cell-mediated adaptive immune response depends upon the activation of T cells via recognition of antigen in the context of a major histocompatibility complex (MHC) molecule. Although studies have shown that alterations in T cell receptor glycosylation reduces the activation threshold, the data on MHC is far less definitive. Here, we discuss the data on MHC glycosylation and the role the glycans might play during the adaptive host response.
glycosylation; MHC class I; MHC class II; HLA; N-glycan; antigen presentation
The anthrax toxins lethal toxin (LT) and edema toxin (ET), are essential virulence factors produced by B. anthracis. These toxins act during two distinct phases of anthrax infection. During the first, prodromal phase, which is often asymptomatic, anthrax toxins act on cells of the immune system to help the pathogen establish infection. Then, during the rapidly progressing (or fulminant) stage of the disease bacteria disseminate via a hematological route to various target tissues and organs, which are typically highly vascularized. As bacteria proliferate in the bloodstream LT and ET begin to accumulate rapidly reaching a critical threshold level that will cause death even when the bacterial proliferation is curtailed by antibiotics. During this final phase of infection the toxins cause an increase in vascular permeability and a decrease in function of target organs including the heart, spleen, kidney, adrenal gland, and brain. In this review, we examine the various biological effects of anthrax toxins, focusing on the fulminant stage of the disease and on mechanisms by which the two toxins may collaborate to cause cardiovascular collapse. We discuss normal mechanisms involved in maintaining vascular integrity and based on recent studies indicating that LT and ET cooperatively inhibit membrane trafficking to cell-cell junctions we explore several potential mechanisms by which the toxins may achieve their lethal effects. We also summarize the effects of other potential virulence factors secreted by B. anthracis and consider the role of toxic factors in the evolutionarily recent emergence of this devastating disease.
Anthrax; B. anthracis; B. cereus; Lethal Factor (LF); Edema Factor (EF); Macrophage; Dendritic Cell; Myeloid cells; Neutrophil; Vascular endothelium; Mural cells; Cardiac; Exocyst; Rab11; Sec15; Cadherin; Notch; cAMP; MAPKK/MKK/MEK; PKA; EPAC
Increases in colonization with serotypes of Streptococcus pneumoniae not contained within the 7-valent pneumococcal conjugate vaccine (PCV) have been reported among children following introduction. Serotype 6C has emerged as prevalent in nasopharyngeal colonization and acute otitis media (AOM), though it is uncommonly recovered from children with invasive pneumococcal disease. Vaccine serotypes within PCV7 have been replaced by nonvaccine serotypes without significant changes in the overall carriage rate. We hypothesize 1) that serotypes vary in their ability to evade host defenses and establish AOM following colonization and 2) the observed reduction in pneumococcal otitis results from a reduced disease potential by some ‘replacement serotypes’. We compared the capacity of S. pneumoniae serotypes 6C and 19A to produce experimental otitis media (EOM) in a chinchilla model. The proportion of chinchillas that developed culture positive EOM and density of middle ear infection was evaluated. EOM was found in 28/82 (34%) ears challenged with 6C compared to 13/18(72.2%) with 19A [p=0.0003]. When disease due to 6C did occur, it was characterized by lowdensity infection. Our findings demonstrate that challenge with serotype 6C results in EOM less frequently than 19A. These data support the need for greater knowledge regarding differences among serotypes to produce AOM.
Streptococcus pneumoniae; complement; virulence