Host–pathogen interaction between macrophage co-cultures with Staphylococcus aureus biofilms

European Journal of Clinical Microbiology & Infectious Diseases - The ability of Staphylococcus aureus to form biofilms is an important virulence factor. During the infectious process, the...     

Infectious Bursal Disease: A complex host–pathogen interaction

Infectious Bursal Disease (IBD) is caused by a small, non-enveloped virus, highly resistant in the outside environment. Infectious Bursal Disease Virus (IBDV) targets the chicken’s immune system in a very comprehensive and complex manner by destroying B lymphocytes, attracting T cells and activating macrophages. As an RNA virus, IBDV has a high mutation rate and may thus give rise to viruses with a modified antigenicity or increased virulence, as emphasized during the last decades. The molecular basis of pathogenicity and the exact cause of clinical disease and death are still poorly understood, as it is not clearly related to the severity of the lesions and the extent of the bursal damage. Recent works however, pointed out the role of an exacerbated innate immune response during the early stage of the infection with upregulated production of promediators that will induce a cytokine storm.     

A fine scale phenotype–genotype virulence map of a bacterial pathogen

A large fraction of the genes from sequenced organisms are of unknown function. This limits biological insight, and for pathogenic microorganisms hampers the development of new approaches to battle infections. There is thus a great need for novel strategies that link genotypes to phenotypes for microorganisms. We describe a high-throughput strategy based on the method Tn-seq that can be applied to any genetically manipulatable microorganism. By screening 17 in vitro and two in vivo (carriage and infection) conditions for the pathogen Streptococcus pneumoniae, we create a resource consisting of >1800 interactions that is rich in new genotype–phenotype relationships. We describe genes that are involved in differential carbon source utilization in the host, as well as genes that are involved both in virulence and in resistance against specific in vitro stresses, thereby revealing selection pressures that the pathogen experiences in vivo. We reveal the secondary response to an antibiotic, including a dual role efflux pump also involved in resistance to pH stress. Through genetic-interaction mapping and gene-expression analysis we define the mechanism of attenuation and the regulatory relationship between a two-component system and a core biosynthetic pathway specific to microorganisms. Thus, we have generated a resource that provides detailed insight into the biology and virulence of S. pneumoniae and provided a road map for similar discovery in other microorganisms.An important goal in biology is to understand the relationship between genotype and phenotype. With respect to pathogenic microorganisms, this goal is especially relevant because the lack of understanding about the function of a significant part of the pan-genome (Medini et al. 2008) is hampering the design of novel strategies to battle infectious diseases. Developing high-throughput approaches for non-model organisms that can match genotypes to phenotypes under in vitro and in vivo (infection) conditions is therefore crucial.A reverse genetics approach based on genome-wide ordered arrays of single gene knockouts (Tong et al. 2001; Schuldiner et al 2005) has been applied to several model organisms (Giaever et al. 2004; Lee et al. 2005; Baba et al. 2006; St Onge et al. 2007; de Berardinis et al. 2008; Liu et al. 2008; Kim et al. 2010; Noble et al. 2010). By determining their growth rate or fitness under defined conditions, genotype–phenotype patterns are obtained. A limitation of this approach is that genome-wide knockout libraries are only available for a handful of organisms. Even for model organisms, experiments often remain restricted to a small number of strains, because constructing new knockout arrays is extremely laborious. In order to make both model and non-model organisms accessible to high-throughput phenotypic profiling and genetic interaction mapping, we recently developed the method Tn-seq with which it is possible to determine each gene''s contribution to fitness in a single experiment (van Opijnen et al. 2009).Here, we report a strategy using Tn-seq to generate detailed genotype–phenotype maps of a microorganism. We apply this strategy to the pathogen Streptococcus pneumoniae, a gram-positive bacterial species and commensal of the human nasopharynx. Dissemination of S. pneumoniae from the nasopharynx frequently leads to otitis media or less often to invasive diseases including pneumonia, meningitis, and bacteremia. Antibiotic resistance is on the rise and each year over a million people succumb to invasive infection with S. pneumoniae, making it one of the most important bacteria clinically (Tuomanen et al. 2004; Harboe et al. 2009; Liñares et al. 2010). Here we measured the fitness of mutant libraries in 17 different in vitro conditions and in two in vivo environments in mice, yielding numerous phenotypes that allowed us to study conditional gene essentiality, discover leads for gene function and antibiotic action, and match defined in vitro stress conditions with in vivo colonization and disease states. Besides creating a resource that provides insight into the biology and virulence of this pathogen, we have drawn up a detailed roadmap that can be used to navigate similar discovery in other microorganisms.     

Coronary–aortic interaction during ventricular isovolumic contraction

In earlier work, we suggested that the start of the isovolumic contraction period could be detected in arterial pressure waveforms as the start of a temporary pre-systolic pressure perturbation (AIC_start, start of the Arterially detected Isovolumic Contraction), and proposed the retrograde coronary blood volume flow in combination with a backwards traveling pressure wave as its most likely origin. In this study, we tested this hypothesis by means of a coronary artery occlusion protocol. In six Yorkshire × Landrace swine, we simultaneously occluded the left anterior descending (LAD) and left circumflex (LCx) artery for 5 s followed by a 20-s reperfusion period and repeated this sequence at least two more times. A similar procedure was used to occlude only the right coronary artery (RCA) and finally all three main coronary arteries simultaneously. None of the occlusion protocols caused a decrease in the arterial pressure perturbation in the aorta during occlusion (P > 0.20) nor an increase during reactive hyperemia (P > 0.22), despite a higher deceleration of coronary blood volume flow (P = 0.03) or increased coronary conductance (P = 0.04) during hyperemia. These results show that the pre-systolic aortic pressure perturbation does not originate from the coronary arteries.     

Host–pathogen interactions in epidermolysis bullosa patients colonized with Staphylococcus aureus

Patients with the genetic blistering disease epidermolysis bullosa (EB) often have chronic wounds that can become colonized by different bacteria, especially the opportunistic pathogen Staphylococcus aureus. We therefore determined the S. aureus colonization rates in EB patients from the Netherlands by collecting swabs from their anterior nares, throats and wounds. Within a period of ∼2 years, more than 90% of the sampled chronic wounds of EB patients were found to be colonized by S. aureus. Molecular typing revealed that EB patients were not colonized by a single S. aureus type. Rather the S. aureus population structure in the sampled EB patients mirrored the local S. aureus population structure within the Netherlands. Furthermore, multiple types of S. aureus were found in close proximity to each other within individual chronic wounds, indicating that these S. aureus types are not mutually exclusive. Over time, strong fluctuations in the S. aureus types sampled from individual EB patients were observed. This high exposure to different S. aureus types is apparently reflected by high plasma levels of antistaphylococcal IgG's, especially in patients carrying multiple S. aureus types. It remains to be determined to what extent this strong immune response protects EB patients against serious staphylococcal infections. Lastly, further research is needed to define the impact of staphylococcal colonization of chronic wounds on the development, exacerbation and healing of such wounds in patients with EB.     

The dynamic nature of bacterial surfaces: Implications for metal–membrane interaction

Bacterial envelopes are chemically complex, diverse structures. Chemical and physical influences from cellular microenvironments force lipids, proteins, and sugars to organize dynamically. This constant reorganization serves to maintain compartmentalization and function, but also affects the influence of charged functional groups that drive electrochemical interactions with metal ions. The interactions of metal species with cell walls are of particular interest because (i) metals must be taken up or excluded to maintain cell function, and (ii) electrochemical interactions between charged metals and anionic ligands are inevitable. In this review we explore the associations of metals with metal-reactive ligands found within bacterial envelopes, and outward to include those within biofilm matrics. The mechanisms that underpin metal binding to these ligands have not been well considered with respect to the dynamic organization of the biological structures themselves. Bacteria respond sensitively and rapidly to growth environment with de novo syntheses of chemical constituents, which can impact metal interactions. We discuss causes of membrane chemical variability as observed in laboratory experiments, and offer consequences for this adaptability in natural settings. The structural impacts of metal ion associations with bacterial envelopes are often overlooked. This review explores how dynamic bacterial surface chemistry influences metal binding and, in turn, how metal ions impact membrane organization in laboratory and natural conditions.     

Mutation of alkyl hydroperoxide reductase gene ahpC of Xanthomonas oryzae pv. oryzae affects hydrogen peroxide accumulation during the rice–pathogen interaction

Hydrogen peroxide (H₂O₂) is usually generated by normal aerobic respiration of pathogens and by the host defense response during plant–pathogen interactions. In this study, histochemical localization of H₂O₂ accumulation in rice inoculated with the wild-type strain (PXO99^A) and the gene deletion mutant (ΔahpC) of alkyl hydroperoxide reductase subunit C (AhpC) of Xanthomonas oryzae pv. oryzae (Xoo), the bacterial blight pathogen of rice, was analyzed. The ΔahpC mutant displayed a significant decrease in endogenous H₂O₂ accumulation which was induced by the compensatory increase in H₂O₂ scavenging activity. The change in the bacterial endogenous H₂O₂ level affected the total amount of H₂O₂ accumulation during the interaction with rice plants. These results suggested that Xoo contributes to H₂O₂ accumulation in rice in a compatible interaction, and pathogen-driving H₂O₂ is in association with cell collapse of rice.     

Host–microbiota interaction and intestinal stem cells in chronic inflammation and colorectal cancer

Inflammatory bowel disease (IBD) and colorectal cancer (CRC) are the major diseases of the lower gastrointestinal tract. The intestinal epithelium plays a critical role in the host’s interactions with the large communities of resident luminal bacteria. Epithelial cells recognize the bacterial components via pattern-recognition receptors. Toll-like receptors (TLRs) are a major class of pattern-recognition receptors that are present on intestinal epithelial cells, including putative stem cells. Stem cells are responsible for tissue homeostasis and regeneration after injury including IBD. Stem cells are also implicated in the pathogenesis of CRC. In susceptible individuals, disruption of normal homeostatic balance between the host’s mucosal cells and enteric microflora is believed to result in aberrant immune responses against the resident commensal bacteria, leading to IBD. Microbiological analyses have revealed that the composition and localization of microbiota is altered in CRC and IBD. It is plausible that stem cells directly sense and respond to microbiota. This review aims to summarize the current knowledge on the effect of microbiota and TLR signaling on intestinal stem cells. It also describes how TLR signaling could affect the stem cell regulatory pathways.     

Genotype–phenotype landscapes for immune–pathogen coevolution

Our immune systems constantly coevolve with the pathogens that challenge them, as pathogens adapt to evade our defense responses, with our immune repertoires shifting in turn. These coevolutionary dynamics take place across a vast and high-dimensional landscape of potential pathogen and immune receptor sequence variants. Mapping the relationship between these genotypes and the phenotypes that determine immune–pathogen interactions is crucial for understanding, predicting, and controlling disease. Here, we review recent developments applying high-throughput methods to create large libraries of immune receptor and pathogen protein sequence variants and measure relevant phenotypes. We describe several approaches that probe different regions of the high-dimensional sequence space and comment on how combinations of these methods may offer novel insight into immune–pathogen coevolution.     

Functional variation reflects intra-strain diversity of Staphylococcus aureus small colony variants in the host–pathogen interaction

It is well acknowledged that genetic variation accounts for the intra-species variability in Staphylococcus aureus isolates. Similarly, deficiency in DNA repair and the resulting increase in genomic mutations determine intra-strain variability in S. aureus small colony variants (SCV). The aim of this study was to investigate whether intra-strain diversity would be associated with an alteration of the host–pathogen interaction. To this end, biofilm formation and immune stimulatory capacity were compared in consecutive SCV isolates originating from a single patient. Despite the relatedness of the isolates, the results revealed significant differences in biofilm formation and immune stimulation determined by Toll-like receptor-2 (TLR2) activity. Variation in the extent of biofilm production could be attributed to differences in the expression of protein A (SpA) and agrA. TLR2 activity only partially correlated with these parameters. Although transiently increased functional activity correlated with clinical remission and was abrogated in MRSA superinfection, we can only speculate that changes in the SCV phenotype reflect alterations in the microbial environment and/or treatment. Taken together, our study provides in vivo evidence for the functional consequences of intra-strain variation in S. aureus.     

Muscle–tendon interaction and EMG profiles of world class endurance runners during hopping

The present study examined the muscle–tendon interaction of ten international level Kenyan runners. Ultrasonography and kinematics were applied together with EMG recordings of lower limb muscles during repetitive hopping performed at maximal level. The ten Kenyans had longer gastro Achilles tendon at rest (p < 0.01) as compared with ten control subjects matched in height. Conversely, the stretching and shortening amplitudes of the tendinous tissues of the medial gastrocnemius (MG) muscle were significantly smaller in the Kenyans than in controls during the contact phase of hopping. This applied also to the fascicle length changes, which were smaller and more homogeneous among Kenyans. These limited musculo-tendinous changes resulted in higher maximal hopping height and in larger power despite their reduced body weight. The associated finding of a greater shortening to stretching ratio of the MG tendinous tissues during contact could imply that the Kenyan MG muscle–tendon unit is optimized to favor efficient storage and recoil of elastic energy, while operating at optimal muscle fascicle working range (plateau region).     

Visual–vestibular interaction during goal directed locomotion: effects of aging and blurring vision

Normal vision overrides perturbed vestibular information for the optimization of performance during goal directed locomotion, suggesting down-regulation of vestibular gain. However, it is not known if the responses to vestibular perturbation are accentuated when vision is impaired. Furthermore, both visual and vestibular systems deteriorate with age. It is not clear, however, how age-related decline in these sensory systems influences visual–vestibular interaction. Therefore, the dual purpose of the present study was to investigate the effects of aging and blurring vision, that simulated the consequences of cataracts, on visual–vestibular interaction. Young and healthy elderly walked to a target located straight ahead with either normal or blurring vision. On randomly selected trials vestibular system perturbation was achieved by applying transmastoidal galvanic vestibular stimulation (GVS). Two different galvanic stimulation intensities were used to provide insight into scaling effect of vestibular perturbation on locomotor performance and how age and vision influences this scaling effect. Maximum path deviation, frontal trunk tilt and postural coordination in the mediolateral direction were evaluated. The magnitude of the path deviation and the trunk tilt response were scaled to the magnitude of the vestibular perturbation in older adults independent of the visual condition. Older participants demonstrated increased coupling of the head and trunk segments irrespective of visual and vestibular perturbations. The results suggest that when visual information was available, the vestibular input reweighting was less effective in older individuals, as shown by the scaled responses to the GVS intensities and the inability to converge efficiently towards the target.     

The response of the host microcirculation to bacterial sepsis: does the pathogen matter?

Sepsis results from the interaction between a host and an invading pathogen. The microcirculatory dysfunction is now considered central in the development of the often deadly multiple organ dysfunction syndrome in septic shock patients. The microcirculatory flow shutdown and flow shunting leading to oxygen demand and supply mismatch at the cellular level and the local activation of inflammatory pathways resulting from the leukocyte–endothelium interactions are both features of the sepsis-induced microcirculatory dysfunction. Although the host response through the inflammatory and immunologic response appears to be critical, there are also evidences that Gram-positive and Gram-negative bacteria can exert different effects at the microcirculatory level. In this review we discuss available data on the potential bacterial-specific microcirculatory alterations observed during sepsis.