Exposure of neutralizing epitopes in the carboxyl-terminal domain of TcdB is altered by a proximal hypervariable region.

The sequence, activity, and antigenicity of TcdB varies between different strains of Clostridium difficile. As a result, ribotype-specific forms of TcdB exhibit different toxicities and are not strongly cross-neutralized. Using a combination of biochemical and immunological approaches, we compared two important variants of TcdB (TcdB012 and TcdB027) to identify the mechanisms through which sequence differences alter epitopes and activity of the toxin. These analyses led to the discovery of a critical variation in the 1753-1851 (B2') region of TcdB, which affects the exposure of neutralizing epitopes in the toxin. Sequence comparisons found that the B2' region exhibits only 77% identity and is the most variable sequence between the two forms of TcdB. A combination of biochemical, analytical, and mutagenesis experiments revealed that the B2' region promotes protein-protein interactions. These interactions appear to shield neutralizing epitopes that would otherwise be exposed in the toxin, an event found to be less prominent in TcdB012 due to sequence differences in the 1773-1780 and 1791-1798 regions of the B2' domain. When the carboxyl-terminal domains of TcdB012 and TcdB027 are swapped, neutralization experiments suggest that the amino terminus of TcdB interacts with the B2' region and impacts the exposure of neutralizing epitopes in the carboxyl terminus. Collectively, these data suggest that variations in the B2' region affect protein-protein interactions within TcdB and that these interactions influence the exposure of neutralizing epitopes.

Stripe-rearing changes multiple aspects of the structure of primary visual cortex.

An important example of brain plasticity is the change in the structure of the orientation map in mammalian primary visual cortex in response to a visual environment consisting of stripes of one orientation. In principle there are many different ways in which the structure of a normal map could change to accommodate increased preference for one orientation. However, until now these changes have been characterised only by the relative sizes of the areas of primary visual cortex representing different orientations. Here we extend to the stripe-reared case a recently proposed Bayesian method for reconstructing orientation maps from intrinsic signal optical imaging data. We first formulated a suitable prior for the stripe-reared case, and developed an efficient method for maximising the marginal likelihood of the model in order to determine the optimal parameters. We then applied this to a set of orientation maps from normal and stripe-reared cats. This analysis revealed that several parameters of overall map structure, specifically the difference between wavelength, scaling and mean of the two vector components of maps, changed in response to stripe-rearing, which together give a more nuanced assessment of the effect of rearing condition on map structure than previous measures. Overall this work expands our understanding of the effects of the environment on brain structure.

Sparse coding can predict primary visual cortex receptive field changes induced by abnormal visual input.

Receptive fields acquired through unsupervised learning of sparse representations of natural scenes have similar properties to primary visual cortex (V1) simple cell receptive fields. However, what drives in vivo development of receptive fields remains controversial. The strongest evidence for the importance of sensory experience in visual development comes from receptive field changes in animals reared with abnormal visual input. However, most sparse coding accounts have considered only normal visual input and the development of monocular receptive fields. Here, we applied three sparse coding models to binocular receptive field development across six abnormal rearing conditions. In every condition, the changes in receptive field properties previously observed experimentally were matched to a similar and highly faithful degree by all the models, suggesting that early sensory development can indeed be understood in terms of an impetus towards sparsity. As previously predicted in the literature, we found that asymmetries in inter-ocular correlation across orientations lead to orientation-specific binocular receptive fields. Finally we used our models to design a novel stimulus that, if present during rearing, is predicted by the sparsity principle to lead robustly to radically abnormal receptive fields.

Ocular Bacterial Infections: A Ten-Year Survey and Review of Causative Organisms Based on the Oklahoma Experience.

Ocular infections can be medical emergencies that result in permanent visual impairment or blindness and loss of quality of life. Bacteria are a major cause of ocular infections. Effective treatment of ocular infections requires knowledge of which bacteria are the likely cause of the infection. This survey of ocular bacterial isolates and review of ocular pathogens is based on a survey of a collection of isolates banked over a ten-year span at the Dean McGee Eye Institute in Oklahoma. These findings illustrate the diversity of bacteria isolated from the eye, ranging from common species to rare and unique species. At all sampled sites, staphylococci were the predominant bacteria isolated. Pseudomonads were the most common Gram-negative bacterial isolate, except in vitreous, where Serratia was the most common Gram-negative bacterial isolate. Here, we discuss the range of ocular infections that these species have been documented to cause and treatment options for these infections. Although a highly diverse spectrum of species has been isolated from the eye, the majority of infections are caused by Gram-positive species, and in most infections, empiric treatments are effective.

Therapeutic potential of Bacillus phage lysin PlyB in ocular infections.

Bacteriophage lytic enzymes (i.e., phage lysins) are a trending alternative for general antibiotics to combat growing antimicrobial resistance. Gram-positive Bacillus cereus causes one of the most severe forms of intraocular infection, often resulting in complete vision loss. It is an inherently ß-lactamase-resistant organism that is highly inflammogenic in the eye, and antibiotics are not often beneficial as the sole therapeutic option for these blinding infections. The use of phage lysins as a treatment for B. cereus ocular infection has never been tested or reported. In this study, the phage lysin PlyB was tested in vitro, demonstrating rapid killing of vegetative B. cereus but not its spores. PlyB was also highly group specific and effectively killed the bacteria in various bacterial growth conditions, including ex vivo rabbit vitreous (Vit). Furthermore, PlyB demonstrated no cytotoxic or hemolytic activity toward human retinal cells or erythrocytes and did not trigger innate activation. In in vivo therapeutic experiments, PlyB was effective in killing B. cereus when administered intravitreally in an experimental endophthalmitis model and topically in an experimental keratitis model. In both models of ocular infection, the effective bactericidal property of PlyB prevented pathological damage to ocular tissues. Thus, PlyB was found to be safe and effective in killing B. cereus in the eye, greatly improving an otherwise devastating outcome. Overall, this study demonstrates that PlyB is a promising therapeutic option for B. cereus eye infections.IMPORTANCEEye infections from antibiotic-resistant Bacillus cereus are devastating and can result in blindness with few available treatment options. Bacteriophage lysins are an alternative to conventional antibiotics with the potential to control antibiotic-resistant bacteria. This study demonstrates that a lysin called PlyB can effectively kill B. cereus in two models of B. cereus eye infections, thus treating and preventing the blinding effects of these infections.

Sparse coding on the spot: spontaneous retinal waves suffice for orientation selectivity.

Ohshiro, Hussain, and Weliky (2011) recently showed that ferrets reared with exposure to flickering spot stimuli, in the absence of oriented visual experience, develop oriented receptive fields. They interpreted this as refutation of efficient coding models, which require oriented input in order to develop oriented receptive fields. Here we show that these data are compatible with the efficient coding hypothesis if the influence of spontaneous retinal waves is considered. We demonstrate that independent component analysis learns predominantly oriented receptive fields when trained on a mixture of spot stimuli and spontaneous retinal waves. Further, we show that the efficient coding hypothesis provides a compelling explanation for the contrast between the lack of receptive field changes seen in animals reared with spot stimuli and the significant cortical reorganisation observed in stripe-reared animals.

The combinatorics of neurite self-avoidance.

During neural development in Drosophila, the ability of neurite branches to recognize whether they are from the same or different neurons depends crucially on the molecule Dscam1. In particular, this recognition depends on the stochastic acquisition of a unique combination of Dscam1 isoforms out of a large set of possible isoforms. To properly interpret these findings, it is crucial to understand the combinatorics involved, which has previously been attempted only using stochastic simulations for some specific parameter combinations. Here we present closed-form solutions for the general case. These reveal the relationships among the key variables and how these constrain possible biological scenarios.

Natural scene statistics and the structure of orientation maps in the visual cortex.

Visual activity after eye-opening influences feature map structure in primary visual cortex (V1). For instance, rearing cats in an environment of stripes of one orientation yields an over-representation of that orientation in V1. However, whether such changes also affect the higher-order statistics of orientation maps is unknown. A statistical bias of orientation maps in normally raised animals is that the probability of the angular difference in orientation preference between each pair of points in the cortex depends on the angle of the line joining those points relative to a fixed but arbitrary set of axes. Natural images show an analogous statistical bias; however, whether this drives the development of comparable structure in V1 is unknown. We examined these statistics for normal, stripe-reared and dark-reared cats, and found that the biases present were not consistently related to those present in the input, or to genetic relationships. We compared these results with two computational models of orientation map development, an analytical model and a Hebbian model. The analytical model failed to reproduce the experimentally observed statistics. In the Hebbian model, while orientation difference statistics could be strongly driven by the input, statistics similar to those seen in experimental maps arose only when symmetry breaking was allowed to occur spontaneously. These results suggest that these statistical biases of orientation maps arise primarily spontaneously, rather than being governed by either input statistics or genetic mechanisms.

Bacterial endophthalmitis: therapeutic challenges and host-pathogen interactions.

Endophthalmitis is an infection of the posterior segment of the eye that frequently results in loss of vision. This devastating result occurs despite prompt and often aggressive therapeutic and surgical intervention. Over the past decade, research has centered on determining the bacterial and host factors involved in this potentially blinding disease. The initial focus on the bacterial factors responsible for intraocular virulence has recently expanded into analysis the inflammatory response to infection, including the molecular and cellular interactions between the pathogen and host. This review discusses the epidemiology and therapeutic challenges posed by endophthalmitis, as well as recent findings from the analysis of interactions between the host and pathogen. Based on these findings, a model for the pathogenesis of endophthalmitis is presented. A more comprehensive understanding of the molecular and cellular interactions taking place between pathogen and host during endophthalmitis will expose possible therapeutic targets designed to arrest the infection and prevent vision loss.

On the simulation of enzymatic digest patterns: the fragmentation of oligomeric and polymeric galacturonides by endo-polygalacturonase II.

A simulation methodology for predicting the time-course of enzymatic digestions is described. The model is based solely on the enzyme's subsite architecture and concomitant binding energies. This allows subsite binding energies to be used to predict the evolution of the relative amounts of different products during the digestion of arbitrary mixtures of oligomeric or polymeric substrates. The methodology has been specifically demonstrated by studying the fragmentation of a population of oligogalacturonides of varying degrees of polymerization, when digested by endo-polygalacturonase II (endo-PG II) from Aspergillus niger.

Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry.

Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-terminal sequence differences to TcdB activity by comparing the binding, uptake, and endosomal localization of TcdB1 and TcdB2 or selected recombinant fragments of these proteins. Our findings suggest that sequence differences in the amino acid 1753 to 1851 region proximal to the combined repetitive oligopeptide domain (CROP) support enhanced uptake of TcdB2 and localization of toxin in acidified endosomes. In the absence of this region, the CROP domains of both forms of the toxin exhibited similar levels of cell interaction, while the addition of amino acids 1753 to 1851 greatly increased toxin binding by only TcdB2. Moreover, the amino acid 1753 to 2366 fragment of TcdB2, but not TcdB1, accumulated to detectable levels in acidified endosomes. Unexpectedly, we discovered an unusual relationship between endocytosis and the efficiency of cell binding for TcdB1 and TcdB2 wherein inhibition of endocytosis by a chemical inhibitor or incubation at a low temperature resulted in a dramatic reduction in cell binding. These findings provide information on sequence variations that may contribute to differences in TcdB1 and TcdB2 toxicity and reveal a heretofore unknown connection between endocytosis and cell binding for this toxin. IMPORTANCE TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2's heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB's variant forms and their mechanisms of cell entry.

Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB.

Clostridium difficile infection (CDI) is a major cause of hospital-associated, antibiotic-induced diarrhea, which is largely mediated by the production of two large multidomain clostridial toxins, TcdA and TcdB. Both toxins coordinate the action of specific domains to bind receptors, enter cells, and deliver a catalytic fragment into the cytosol. This results in GTPase inactivation, actin disassembly, and cytotoxicity. TcdB in particular has been shown to encode a region covering amino acids 1753 to 1851 that affects epitope exposure and cytotoxicity. Surprisingly, studies here show that several peptides derived from this region, which share the consensus sequence 1769NVFKGNTISDK1779, protect cells from the action of TcdB. One peptide, PepB2, forms multiple interactions with the carboxy-terminal region of TcdB, destabilizes TcdB structure, and disrupts cell binding. We further show that these effects require PepB2 to form a higher-order polymeric complex, a process that requires the central GN amino acid pair. These data suggest that TcdB1769-1779 interacts with repeat sequences in the proximal carboxy-terminal domain of TcdB (i.e., the CROP domain) to alter the conformation of TcdB. Furthermore, these studies provide insights into TcdB structure and functions that can be exploited to inactivate this critical virulence factor and ameliorate the course of CDI.IMPORTANCEClostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment.

TLR4 contributes to the host response to Klebsiella intraocular infection.

PURPOSE/AIM: Klebsiella pneumoniae causes a blinding infection called endogenous endophthalmitis. The role of innate immune recognition of K. pneumoniae in the eye during infection is not known. We hypothesized that intraocular recognition of K. pneumoniae was mediated by Toll-like receptor (TLR)-4 and may be dependent on MagA-regulated hypermucoviscosity. MATERIALS AND METHODS: Experimental endophthalmitis was induced in C57BL/6J or TLR4(-/-) mice by intravitreal injection of 100 CFU of wild type or ΔmagA K. pneumoniae. Infection and inflammation were quantified by determining viable K. pneumoniae per eye, retinal responses via electroretinography, myeloperoxidase activity of infiltrating neutrophils and the proinflammatory cytokine and chemokine response. RESULTS: C57BL/6J and TLR4(-/-) mice could not control intraocular wild-type K. pneumoniae growth. TLR4(-/-) mice were less able than C57BL/6J to control the intraocular growth of ΔmagA K. pneumoniae. Retinal function testing suggested that infection with ΔmagA K. pneumoniae resulted in less retinal function loss. There was a TLR4-dependent delay in initial neutrophil recruitment, regardless of the infecting organism. The proinflammatory cytokine/chemokine data supported these results. These findings were not due to an inability of TLR4(-/-) neutrophils to recognize or kill K. pneumoniae. CONCLUSIONS: These studies suggest that TLR4 is important in the early intraocular recognition and host response to K. pneumoniae. However, the role of MagA in TLR4-mediated intraocular recognition and subsequent inflammation is less clear.

Variations in virulence and molecular biology among emerging strains of Clostridium difficile.

Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile, now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade.

Randomly oriented edge arrangements dominate naturalistic arrangements in binocular rivalry.

Biological visual systems are highly adapted to the image statistics of the natural world. A particularly important aspect of the statistics of natural scenes is the arrangements of edges they contain. Here, we examined how different arrangements of edges influence human perceptual saliency using a binocular rivalry paradigm. We constructed fields of randomly positioned Gabor patches with orientation arrangements containing co-oriented, co-circular and naturalistic structure. We rivalled these against arrangements with random orientations, which have higher entropy. Surprisingly, we found that fields with randomly oriented edges consistently dominated over the more ordered arrangements. These results suggest that visual scene entropy may be a key variable in early perceptual saliency.

MHC class II and non-MHC class II genes differentially influence humoral immunity to Bacillus anthracis lethal factor and protective antigen.

Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class II versus non-MHC class II genes in the humoral response to PA and LF immunization using three immunized strains of inbred mice: A/J (H-2k at the MHC class II locus), B6 (H-2b), and B6.H2k (H-2k). IgG antibody titers to LF were controlled primarily by the MHC class II locus, whereas IgG titers to PA were strongly influenced by the non-MHC class II genetic background. Conversely, the humoral fine specificity of reactivity to LF appeared to be controlled primarily through non-MHC class II genes, while the specificity of reactivity to PA was more dependent on MHC class II. Common epitopes, reactive in all strains, occurred in both LF and PA responses. These results demonstrate that MHC class II differentially influences humoral immune responses to LF and PA.

Contribution of mucoviscosity-associated gene A (magA) to virulence in experimental Klebsiella pneumoniae endophthalmitis.

PURPOSE: Endogenous endophthalmitis secondary to Klebsiella pneumoniae liver abscess is a blinding infection that is being reported more frequently in the literature. The K1 capsule and magA contribute to virulence of systemic infection in mice; however, little is known about the role of magA in secondary ocular infections. METHODS: To assess the role of K. pneumoniae capsule in endophthalmitis, the authors induced experimental endophthalmitis by direct inoculation of 100 colony-forming unit wild-type, magA-deficient, or magA-complemented K. pneumoniae into the posterior segments of mouse eyes. Eyes were analyzed by quantitation of viable bacteria, retinal function, and inflammatory cell influx as well as by histology. RESULTS: Wild-type K1 K. pneumoniae caused significant ocular disease. At the end point of 24 hours postinfection, eyes infected with wild-type K. pneumoniae retained significantly less retinal A-wave function than eyes infected with an isogenic magA-mutant strain. B-wave function retention was also greater in eyes infected with the magA mutant than with wild-type K. pneumoniae. Additionally, intraocular growth of the magA-deficient strain was less than it was in the wild-type strain. The amount of myeloperoxidase elicited was also significantly higher for wild-type-infected eyes at 24 hours. CONCLUSIONS: These results indicate that in the eye, the K1 capsule of invasive K. pneumoniae significantly contributes to the ability of the bacteria to disrupt retinal function, to grow to high density, and to persist despite immune cell recruitment.

Statistical structure of lateral connections in the primary visual cortex.

BACKGROUND: The statistical structure of the visual world offers many useful clues for understanding how biological visual systems may understand natural scenes. One particularly important early process in visual object recognition is that of grouping together edges which belong to the same contour. The layout of edges in natural scenes have strong statistical structure. One such statistical property is that edges tend to lie on a common circle, and this 'co-circularity' can predict human performance at contour grouping. We therefore tested the hypothesis that long-range excitatory lateral connections in the primary visual cortex, which are believed to be involved in contour grouping, display a similar co-circular structure. RESULTS: By analyzing data from tree shrews, where information on both lateral connectivity and the overall structure of the orientation map was available, we found a surprising diversity in the relevant statistical structure of the connections. In particular, the extent to which co-circularity was displayed varied significantly. CONCLUSIONS: Overall, these data suggest the intriguing possibility that V1 may contain both co-circular and anti-cocircular connections.

Hypermucoviscosity as a virulence factor in experimental Klebsiella pneumoniae endophthalmitis.

PURPOSE: Klebsiella pneumoniae is a common cause of endogenous bacterial endophthalmitis, a disease that frequently results in a poor visual outcome. Hypermucoviscosity has been identified as a virulence factor among clinical bacteremia isolates of K. pneumoniae. In this study, an experimental murine model of K. pneumoniae endophthalmitis was established, and the role of hypermucoviscosity in its pathogenesis was analyzed. METHODS: C57BL/6J mice were intravitreously injected with 100 CFU of hypermucoviscous (HMV+) or nonhypermucoviscous (HMV-) K. pneumoniae. Intraocular bacterial growth, retinal function, gross pathology, and inflammatory responses were monitored every 3 hours until the eyes lost significant (>90%) retinal function, or the infection appeared to clear. RESULTS: The HMV+ strain grew logarithmically in eyes until approximately 15 hours postinfection (PI), reaching a stationary phase of growth at approximately 8.0 log(10) CFU/eye. The HMV- strain grew logarithmically to approximately 7.6 log(10) by 18 hours, but bacterial count declined to approximately 6.4 log(10) CFU/eye by 21 hours PI. Eyes infected with the HMV+ strain retained approximately 35% a-wave and <10% b-wave function by 18 hours PI. These eyes also had a cumulative clinical score of 14+ by 18 hours and underwent phthisis between 21 and 24 hours. Eyes infected with the HMV- strain had a cumulative clinical score of <6 and retained >60% a-wave and >50% b-wave function throughout 21 hours. Five of 7 eyes had <100 CFU HMV- K. pneumoniae at 27 hours PI. CONCLUSIONS: The findings demonstrate the site-threatening consequences of K. pneumoniae endophthalmitis and the importance of the hypermucoviscosity phenotype in the pathogenesis of experimental K. pneumoniae endophthalmitis.