SmC2P1, a C2 domain protein from Scophthalmus maximus that binds bacterial pathogen-infected lymphocytes and reduces bacterial survival

C2 domains are protein structural modules found in many eukaryotic proteins involved in signal transduction, membrane trafficking, and immune defense. Most of the studied C2 domain-containing proteins are multi-domained in structure, in which the C2 domain is an independently folded motif and plays an essential role in calcium-dependent membrane-targeting. Although C2 domains isolated from intact proteins have been studied for biological functions, no study on natural proteins containing C2 domain only has been documented. In this study, we identified a Scophthalmus maximus protein SmC2P1 that is comprised of a single C2 domain and lacks any other apparent domain structures. The deduced amino acid sequence of SmC2P1 contains 129 residues and shares 36-38% identities with the C2 domains of the perforins of several fish species. Like typical C2 domains, SmC2P1 is predicted to organize into eight beta-strands with a Ca2+-binding site located in inter-strand loops. SmC2P1 expression was detected, in deceasing order, in liver, spleen, blood, brain, muscle, kidney, gill, and heart. Experimental challenge of turbot with a bacterial pathogen significantly upregulated SmC2P1 expression in kidney in a time-dependent manner. Recombinant SmC2P1 purified from yeast exhibits no hemolytic activity but binds to pathogen-infected kidney lymphocytes in the presence of calcium. Furthermore, interaction of recombinant SmC2P1 with bacterium-infected lymphocytes reduced bacterial survival. These results indicate that SmC2P1 is a functional protein that is involved in host immune defense against bacterial infection. (C) 2010 Elsevier Ltd. All rights reserved.

Impact of cigarette smoke exposure on host-bacterial pathogen interactions

The human respiratory tract of individuals with normal lung function maintains a fine-tuned balance, being asymptomatically colonised by the normal microbiota in the upper airways and sterile in the lower tract. This equilibrium may be disrupted by the exposure to insults such as cigarette smoke. In the respiratory tract, the complex and noxious nature of inhaled cigarette smoke alters host-microorganisminteraction dynamics at all anatomical levels, causing infections in many cases. Moreover, continuous exposure to cigarette smoke itself causes deleterious effects on the host that can trigger the development of chronic respiratory diseases, such as chronic obstructive pulmonary disease (COPD) and lung cancer. COPD is an irreversible airflow obstruction associated with emphysema, fibrosis, mucus hypersecretion and persistent colonisation of the lower airways by opportunistic pathogens. COPD patients keep a stable (without exacerbation) but progressively worsening condition and suffer periodic exacerbations caused, in most cases, by infections. Although smoking and smoking-associated diseases are associated with a high risk of infection, most therapies aim to reduce inflammatory parameters, but do not necessarily take into account the presence of persistent colonisers. The effect of cigarette smoke on host-pathogen interaction dynamics in the respiratory tract, together with current and novel therapies, is discussed. Copyright©ERS 2012.

Bacterial pathogen evolution: breaking news

The immense social and economic impact of bacterial pathogens, from drug-resistant infections in hospitals to the devastation of agricultural resources, has resulted in major investment to understand the causes and conse- quences of pathogen evolution. Recent genome se- quencing projects have provided insight into the evolution of bacterial genome structures; revealing the impact of mobile DNA on genome restructuring and pathogenicity. Sequencing of multiple genomes of relat- ed strains has enabled the delineation of pathogen evo- lution and facilitated the tracking of bacterial pathogens globally. Other recent theoretical and empirical studies have shown that pathogen evolution is significantly influenced by ecological factors, such as the distribution of hosts within the environment and the effects of co- infection. We suggest that the time is ripe for experi- mentalists to use genomics in conjunction with evolu- tionary ecology experiments to further understanding of how bacterial pathogens evolve.

The influence of the accessory genome on bacterial pathogen evolution

Bacterial pathogens exhibit significant variation in their genomic content of virulence factors. This reflects the abundance of strategies pathogens evolved to infect host organisms by suppressing host immunity. Molecular arms-races have been a strong driving force for the evolution of pathogenicity, with pathogens often encoding overlapping or redundant functions, such as type III protein secretion effectors and hosts encoding ever more sophisticated immune systems. The pathogens’ frequent exposure to other microbes, either in their host or in the environment, provides opportunities for the acquisition or interchange of mobile genetic elements. These DNA elements accessorise the core genome and can play major roles in shaping genome structure and altering the complement of virulence factors. Here, we review the different mobile genetic elements focusing on the more recent discoveries and highlighting their role in shaping bacterial pathogen evolution.

The causative pathogen determines the inflammatory profile in cerebrospinal fluid and outcome in patients with bacterial meningitis

BACKGROUND The brain's inflammatory response to the infecting pathogen determines the outcome of bacterial meningitis (BM), for example, the associated mortality and the extent of brain injury. The inflammatory cascade is initiated by the presence of bacteria in the cerebrospinal fluid (CSF) activating resident immune cells and leading to the influx of blood derived leukocytes. To elucidate the pathomechanisms behind the observed difference in outcome between different pathogens, we compared the inflammatory profile in the CSF of patients with BM caused by Streptococcus pneumonia (n = 14), Neisseria meningitidis (n = 22), and Haemophilus influenza (n = 9). METHODS CSF inflammatory parameters, including cytokines and chemokines, MMP-9, and nitric oxide synthase activity, were assessed in a cohort of patients with BM from Burkina Faso. RESULTS Pneumococcal meningitis was associated with significantly higher CSF concentrations of IFN-γ , MCP-1, and the matrix-metalloproteinase (MMP-) 9. In patients with a fatal outcome, levels of TNF-α, IL-1 β, IL-1RA, IL-6, and TGF-α were significantly higher. CONCLUSION The signature of pro- and anti-inflammatory mediators and the intensity of inflammatory processes in CSF are determined by the bacterial pathogen causing bacterial meningitis with pneumococcal meningitis being associated with a higher case fatality rate than meningitis caused by N. meningitidis or H. influenzae.

NDR1, a locus of Arabidopsis thaliana that is required for disease resistance to both a bacterial and a fungal pathogen.

We have employed Arabidopsis thaliana as a model host plant to genetically dissect the molecular pathways leading to disease resistance. A. thaliana accession Col-0 is susceptible to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 but resistant in a race-specific manner to DC3000 carrying any one of the cloned avirulence genes avrB, avrRpm1, avrRpt2, and avrPph3. Fast-neutron-mutagenized Col-0 M2 seed was screened to identify mutants susceptible to DC3000(avrB). Disease assays and analysis of in planta bacterial growth identified one mutant, ndr1-1 (nonrace-specific disease resistance), that was susceptible to DC3000 expressing any one of the four avirulence genes tested. Interestingly, a hypersensitive-like response was still induced by several of the strains. The ndr1-1 mutation also rendered the plant susceptible to several avirulent isolates of the fungal pathogen Peronospora parasitica. Genetic analysis of ndr1-1 demonstrated that the mutation segregated as a single recessive locus, located on chromosome III. Characterization of the ndr1-1 mutation suggests that a common step exists in pathways of resistance to two unrelated pathogens.

Does a 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine prevent respiratory exacerbations in children with recurrent protracted bacterial bronchitis, chronic suppurative lung disease and bronchiectasis : protocol for a randomised controlled trial

Background Recurrent protracted bacterial bronchitis (PBB), chronic suppurative lung disease (CSLD) and bronchiectasis are characterised by a chronic wet cough and are important causes of childhood respiratory morbidity globally. Haemophilus influenzae and Streptococcus pneumoniae are the most commonly associated pathogens. As respiratory exacerbations impair quality of life and may be associated with disease progression, we will determine if the novel 10-valent pneumococcal-Haemophilus influenzae protein D conjugate vaccine (PHiD-CV) reduces exacerbations in these children. Methods A multi-centre, parallel group, double-blind, randomised controlled trial in tertiary paediatric centres from three Australian cities is planned. Two hundred six children aged 18 months to 14 years with recurrent PBB, CSLD or bronchiectasis will be randomised to receive either two doses of PHiD-CV or control meningococcal (ACYW(135)) conjugate vaccine 2 months apart and followed for 12 months after the second vaccine dose. Randomisation will be stratified by site, age (<6 years and >= 6 years) and aetiology (recurrent PBB or CSLD/bronchiectasis). Clinical histories, respiratory status (including spirometry in children aged >= 6 years), nasopharyngeal and saliva swabs, and serum will be collected at baseline and at 2, 3, 8 and 14 months post-enrolment. Local and systemic reactions will be recorded on daily diaries for 7 and 30 days, respectively, following each vaccine dose and serious adverse events monitored throughout the trial. Fortnightly, parental contact will help record respiratory exacerbations. The primary outcome is the incidence of respiratory exacerbations in the 12 months following the second vaccine dose. Secondary outcomes include: nasopharyngeal carriage of H. influenzae and S. pneumoniae vaccine and vaccine-related serotypes; systemic and mucosal immune responses to H. influenzae proteins and S. pneumoniae vaccine and vaccine-related serotypes; impact upon lung function in children aged >= 6 years; and vaccine safety. Discussion As H. influenzae is the most common bacterial pathogen associated with these chronic respiratory diseases in children, a novel pneumococcal conjugate vaccine that also impacts upon H. influenzae and helps prevent respiratory exacerbations would assist clinical management with potential short- and long-term health benefits. Our study will be the first to assess vaccine efficacy targeting H. influenzae in children with recurrent PBB, CSLD and bronchiectasis.

Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids.

Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen.

Capsular sialic acid of Streptococcus suis serotype 2 binds to swine influenza virus and enhances bacterial interactions with virus-infected tracheal epithelial cells.

Streptococcus suis serotype 2 is an important swine bacterial pathogen, and it is also an emerging zoonotic agent. It is unknown how S. suis virulent strains, which are usually found in low quantities in pig tonsils, manage to cross the first host defense lines to initiate systemic disease. Influenza virus produces a contagious infection in pigs which is frequently complicated by bacterial coinfections, leading to significant economic impacts. In this study, the effect of a preceding swine influenza H1N1 virus (swH1N1) infection of swine tracheal epithelial cells (NTPr) on the ability of S. suis serotype 2 to adhere to, invade, and activate these cells was evaluated. Cells preinfected with swH1N1 showed bacterial adhesion and invasion levels that were increased more than 100-fold compared to those of normal cells. Inhibition studies confirmed that the capsular sialic acid moiety is responsible for the binding to virus-infected cell surfaces. Also, preincubation of S. suis with swH1N1 significantly increased bacterial adhesion to/invasion of epithelial cells, suggesting that S. suis also uses swH1N1 as a vehicle to invade epithelial cells when the two infections occur simultaneously. Influenza virus infection may facilitate the transient passage of S. suis at the respiratory tract to reach the bloodstream and cause bacteremia and septicemia. S. suis may also increase the local inflammation at the respiratory tract during influenza infection, as suggested by an exacerbated expression of proinflammatory mediators in coinfected cells. These results give new insight into the complex interactions between influenza virus and S. suis in a coinfection model.

Abscisic acid regulation of plant defence responses during pathogen attack

The plant hormone, abscisic acid (ABA), has previously been shown to have an impact on the resistance or susceptibility of plants to pathogens. In this thesis, it was shown that ABA had a regulatory effect on an extensive array of plant defence responses in three different plant and pathogen interaction combinations as well as following the application of an abiotic elicitor. In unique studies using ABA deficient mutants of Arabidopsis, exogenous ABA addition or ABA biosynthesis inhibitor application and simulated drought stress, ABA was shown to have a profound effect on the outcome of interactions between plants and pathogens of differing lifestyles and from different kingdoms. The systems used included a model plant and an important agricultural species: Arabidopsis thaliana (Arabidopsis) and Peronospora parasitica (a biotrophic Oomycete pathogen), Arabidopsis and Pseudomonas syringae pathovar tomato (a biotrophic bacterial pathogen) and an unrelated plant species, soybean (Glycine max) and Phytophthora sojae (a hemibiotrophic Oomycete pathogen), Generally, a higher than basal endogenous ABA concentration within plant tissues at the time of avirulent pathogen inoculation, caused an interaction shift towards what phenotypically resembled susceptibility. Conversely, a lower than basal endogenous ABA concentration in plants inoculated with a virulent pathogen caused a shift towards resistance. An extensive suppressive effect of ABA on defence responses was revealed by a range of techniques that included histochemical, biochemical and molecular approaches. A universal effect of ABA on suppression or induction of the phenylpropanoid pathway via regulation of the key entry point gene, phenylalanine ammonia-lyase (PAL), when stimulated by biotic or abiotic elicitors was shown. ABA also influenced a wide variety of other defence-related components such as: the development of a hypersensitive response (HR), the accumulation of the reactive oxyden species, hydrogen peroxide and the cell wall strengthening compounds lignin and callose, accumulation of SA and the phytoalexin, glyceollin and the transcription of the SA-dependent pathogenesis- related gene (PR-1). The near genome-wide microarray gene expression analysis of an ABA induced susceptible interaction also revealed an yet unprecedented insight into the great diversity of defence responses that were influenced by ABA that included: disease resistance like proteins, antimicrobial proteins as well as phenylpropanoid and tryptophan pathway enzymes. Subtle differences were found in the number and type of defence responses that were regulated by ABA in each type of plant and pathogen interaction that was studied. This thesis has clearly identified in plant/pathogen interactions previously unknown and important roles for ABA in the regulation of many defence responses.

[In search of strategies for preventing brain damage as a sequela of bacterial meningitis]

Multiplication of bacteria within the central nervous system compartment triggers a host response with an overshooting inflammatory reaction which leads to brain parenchyma damage. Some of the inflammatory and neurotoxic mediators involved in the processes leading to neuronal injury during bacterial meningitis have been identified in recent years. As a result, the therapeutic approach to the disease has widened from eradication of the bacterial pathogen with antibiotics to attenuation of the detrimental effects of host defences. Corticosteroids represent an example of the adjuvant therapeutic strategies aimed at downmodulating excessive inflammation in the infected central nervous system. Pathophysiological concepts derived from an experimental rat model of bacterial meningitis revealed possible therapeutic strategies for prevention of brain damage. The insights gained led to the evaluation of new therapeutic modalities such as anticytokine agents, matrix metalloproteinase inhibitors, antioxidants, and antagonists of endothelin and glutamate. Bacterial meningitis is still associated with persistent neurological sequelae in approximately one third of surviving patients. Future research in the model will evaluate whether the neuroprotective agents identified so far have the potential to attenuate learning disabilities as a long-term consequence of bacterial meningitis.

Host-pathogen interactions of secreted and surface Staphylococcus aureus factors

Staphylococcus aureus is an opportunistic bacterial pathogen that can infect humans and other species. It utilizes an arsenal of virulence factors to cause disease, including secreted and cell wall anchored factors. Secreted toxins attack host cells, and pore-forming toxins destroy target cells by causing cell lysis. S. aureus uses cell-surface adhesins to attach to host molecules thereby facilitating host colonization. The Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are a family of cell-wall anchored proteins that target molecules like fibronectin and fibrinogen. The Serine-aspartate repeat (Sdr) proteins are a subset of staphylococcal MSCRAMMs that share similar domain organization. Interestingly, the amino-terminus, is composed of three immunoglobulin-folded subdomains (N1, N2, and N3) that contain ligand-binding activity. Clumping factors A and B (ClfA and ClfB) and SdrG are Sdr proteins that bind to fibrinogen (Fg), a large, plasma glycoprotein that is activated during the clotting cascade to form fibrin. In addition to recognizing fibrinogen, ClfA and ClfB can bind to other host ligands. Analysis of S. aureus strains that cause osteomyelitis led to the discovery of the bone-sialoprotein-binding protein (Bbp), an Sdr protein. Because several MSCRAMMs target more than one molecule, I hypothesized that Bbp may recognize other host proteins. A ligand screen revealed that the recombinant construct BbpN2N3 specifically recognizes human Fg. Surface plasmon resonance was used to determine the affinity of BbpN2N3 for Fg, and a dissociation constant of 540 nM was determined. Binding experiments performed with recombinant Fg chains were used to map the binding of BbpN2N3 to the Fg Aalpha chain. Additionally, Bbp expressed on the surface of Lactococcus lactis and S. aureus Newman bald mediated attachment of these bacteria to Fg Aalpha. To further characterize the interaction between the two proteins, isothermal titration calorimetry and inhibition assays were conducted with synthetic Fg Aalpha peptides. To determine the physiological implications of Bbp binding to Fg, the effect of Bbp on fibrinogen clotting was studied. Results show that Bbp binding to Fg inhibits the formation of fibrin. The consequences of this interaction are currently under investigation. Together, these data demonstrate that human Fg is a novel ligand for Bbp. This study indicates that the MSCRAMM Bbp may aid in staphylococcal attachment by targeting both an extracellular matrix and a blood plasma protein. The implications of these novel findings are discussed.

A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses

The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His 6 -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1 D299A non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death.

Exploring new roles for the rpoS gene in the survival and virulence of the fire blight pathogen Erwinia amylovora

Erwinia amylovora causes fire blight in economically important plants of the family Rosaceae. This bacterial pathogen spends part of its life cycle coping with starvation and other fluctuating environmental conditions. In many Gram-negative bacteria, starvation and other stress responses are regulated by the sigma factor RpoS. We obtained an E. amylovora rpoS mutant to explore the role of this gene in starvation responses and its potential implication in other processes not yet studied in this pathogen. Results showed that E. amylovora needs rpoS to develop normal starvation survival and viable but nonculturable (VBNC) responses. Furthermore, this gene contributed to stationary phase cross-protection against oxidative, osmotic, and acid stresses and was essential for cross-protection against heat shock, but nonessential against acid shock. RpoS also mediated regulation of motility, exopolysaccharide synthesis, and virulence in immature loquats, but not in pear plantlets, and contributed to E. amylovora survival in nonhost tissues during incompatible interactions. Our results reveal some unique roles for the rpoS gene in E. amylovora and provide new knowledge on the regulation of different processes related to its ecology, including survival in different environments and virulence in immature fruits.

Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the “oxidative burst,” they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.