Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism

Granulomatous amoebic encephalitis due to Acanthamoeba castellanii is a serious human infection with fatal consequences, but it is not clear how the circulating amoebae interact with the blood-brain barrier and transmigrate into the central nervous system. We studied the effects of an Acanthamoeba encephalitis isolate belonging to the T1 genotype on human brain microvascular endothelial cells, which constitute the blood-brain barrier. Using an apoptosis-specific enzyme-linked immunosorbent assay, we showed that Acanthamoeba induces programmed cell death in brain microvascular endothelial cells. Next, we observed that Acanthamoeba specifically activates phosphatidylinositol 3-kinase. Acanthamoeba-mediated brain endothelial cell death was abolished using LY294002, a phosphatidylinositol 3-kinase inhibitor. These results were further confirmed using brain microvascular endothelial cells expressing dominant negative forms of phosphatidylinositol 3-kinase. This is the first demonstration that Acanthamoeba-mediated brain microvascular endothelial cell death is dependent on phosphatidylinositol 3-kinase

Detection of Vibrio cholerae and Acanthamoeba species from same natural water samples collected from different cholera endemic areas in Sudan

<p>Abstract</p> <p>Background</p> <p><it>Vibrio cholerae </it>O1 and <it>V. cholerae </it>O139 infect humans, causing the diarrheal and waterborne disease cholera, which is a worldwide health problem. <it>V. cholerae </it>and the free-living amoebae <it>Acanthamoeba </it>species are present in aquatic environments, including drinking water and it has shown that <it>Acanthamoebae </it>support bacterial growth and survival. Recently it has shown that <it>Acanthamoeba </it>species enhanced growth and survival of <it>V. cholerae </it>O1 and O139. Water samples from different cholera endemic areas in Sudan were collected with the aim to detect both <it>V. cholerae </it>and <it>Acanthamoeba </it>species from same natural water samples by polymerase chain reaction (PCR).</p> <p>Findings</p> <p>For the first time both <it>V. cholerae </it>and <it>Acanthamoeba </it>species were detected in same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected <it>V. cholerae </it>was found with <it>Acanthamoeba </it>in same water samples.</p> <p>Conclusions</p> <p>The current findings disclose <it>Acanthamoedae </it>as a biological factor enhancing survival of <it>V. cholerae </it>in nature.</p

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions

Sequencing illustrates the transcriptional response of Legionella pneumophila during infection and identifies seventy novel small non-coding RNAs

Second generation sequencing has prompted a number of groups to re-interrogate the transcriptomes of several bacterial and archaeal species. One of the central findings has been the identification of complex networks of small non-coding RNAs that play central roles in transcriptional regulation in all growth conditions and for the pathogen’s interaction with and survival within host cells. Legionella pneumophila is a Gram-negative facultative intracellular human pathogen with a distinct biphasic lifestyle. One of its primary environmental hosts in the free-living amoeba Acanthamoeba castellanii and its infection by L. pneumophila mimics that seen in human macrophages. Here we present analysis of strand specific sequencing of the transcriptional response of L. pneumophila during exponential and post-exponential broth growth and during the replicative and transmissive phase of infection inside A. castellanii. We extend previous microarray based studies as well as uncovering evidence of a complex regulatory architecture underpinned by numerous non-coding RNAs. Over seventy new non-coding RNAs could be identified; many of them appear to be strain specific and in configurations not previously reported. We discover a family of non-coding RNAs preferentially expressed during infection conditions and identify a second copy of 6S RNA in L. pneumophila. We show that the newly discovered putative 6S RNA as well as a number of other non-coding RNAs show evidence for antisense transcription. The nature and extent of the non-coding RNAs and their expression patterns suggests that these may well play central roles in the regulation of Legionella spp

Fast Multispectral2Gray

A standard approach to generating a grayscale equivalent to an input multi-spectral image involves calculating the so-called structure tensor at each image pixel. Defining contrast as associated with the maximum-change direction of this matrix, the gray gradient is identified with the first eigenvector direction, with gradient strength given by the square root of its eigenvalue. However, aside from the inherent complexity of such an approach, each pixel’s gradient still possesses a sign ambiguity, since an eigenvector is given only up to a sign. This is ostensibly resolved by looking at how one of the color channels behaves, or how the the luminance changes, or how overall integrability is affected by each sign choice. Instead, we would like to circumvent the sign problem in the first place, and also avoid calculating the costly eigenvector decomposition. We suggest replacing the eigenvector approach by generating a grayscale gradient equal to the maximum gradient amongst the color or multi-spectral channels ’ gradients, in each of x, y. color or But in order not to neglect the tensor approach, we consider the relationship between the complex and the simple approaches. We also note that, at each pixel, we have both forward-facing and backward-facing derivatives, which are different. In a novel approach, we consider a tensor formed from both. Then, over a standard training set, we ask for an optimum set of weights for all the maximum gradients such that the simple maxima scheme generates a grayscale structure tensor to best match the original, multi-spectral, one. If we use only forward-facing derivatives, a fast Fourier-based solution is possible. But instead, we find that a simple scheme that equally weights maxima in the forward-facing and backward-facing directions produces superlative results if a reset step is included, in a spatial-domain solution. Grayscale results are shown to be excellent, and the algorithm is very fast. 1

The role of proteases in the differentiation of Acanthamoeba castellanii.

Proteases are significant determinants of protozoan pathogenicity and cytolysis of host cells. However, there is now growing evidence of their involvement in cellular differentiation. Acanthamoeba castellanii of the T4 genotype elaborates a number of proteases, which are inhibited by the serine protease inhibitor phenylmethylsulphonyl fluoride. Using this and other selective protease inhibitors, in tandem with siRNA primers, specific to the catalytic site of Acanthamoeba serine proteases, we demonstrate that serine protease activity is crucial for the differentiation of A. castellanii. Furthermore, both encystment and excystment of A. castellanii was found to be dependent on serine protease function

Staphylococcus aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells

Staphylococcus aureus is a leading cause of nosocomial infections. Haematogenous spread is a pre-requisite but it is not clear how S. aureus survive the onslaught of macrophages. Acanthamoeba is a protozoan pathogen that is remarkably similar to macrophages, particularly in their cellular structure (morphological and ultra-structural features), molecular motility, biochemical physiology, ability to capture prey by phagocytosis and interactions with microbial pathogens. Thus, we hypothesize that S. aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells. Here, we studied interactions of methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and Staphylococcus epidermidis (SE) with Acanthamoeba castellanii belonging to the T4 genotype and macrophage-like cells (ThP1). The findings revealed that both MRSA and MSSA exhibited similarities in their binding/association and invasion of A. castellanii and ThP1 cells. Long-term incubation showed that MRSA and MSSA can survive intracellularly of both Acanthamoeba and ThP1 cells. Overall, these findings suggest that Acanthamoeba exhibit similar characteristics with ThP1 macrophage-like cells in their interaction with MRSA and MSSA. Additionally it was shown that bacteria survive inside Acanthamoeba during the encystment process as evidenced by bacterial recovery from mature cysts. Given that Acanthamoeba cysts are airborne, these findings suggest that cysts may act as ``Trojan horse'' to help spread MRSA to susceptible hosts. (C) 2012 Elsevier Inc. All rights reserved

Role of human tear fluid in Acanthamoeba interactions with the human corneal epithelial cells

Acanthamoeba keratitis is a painful and progressive sight-threatening infection. However, the precise mechanisms associated with the pathogenesis and pathophysiology of Acanthamoeba keratitis remain incompletely understood. Using tears from healthy individuals and an Acanthamoeba keratitis patient, we demonstrated that both subjects exhibited similar levels of Acanthamoeba-specific IgA as demonstrated by Western blotting and enzyme-linked immunosorbent assays. However, normal tears were slightly more potent in reducing Acanthamoeba binding to human corneal epithelial cells, compared with tears from Acanthamoeba keratitis patient (P>0.05 using paired T-test, one-tail distribution). Neither normal tears nor Acanthamoeba keratitis tears had any protective effects on Acanthamoeba-mediated corneal epithelial cell cytotoxicity. Both lysozyme and lactoferrin which are major constituents of the tear film and possess antibacterial properties exhibited no significant effects on Acanthamoeba binding to and cytotoxicity of human corneal epithelial cells. The role of contact lens wear in Acanthamoeba keratitis is discussed further

Local Linear Models for Improved von Kries Adaptation

In this paper we explore the conditions under which the von Kries model of colour constancy is exact,an investigation motivated by the fact that in practice the model has been shown to work well for a wide range of imaging devices [1]despite the fact that existing theory [2]predicts that it should perform poorly.We present a modified theory which reconciles this apparent contradiction and which is based on the observation that von Kries adaptation treats sensor responses independently of one another.Starting from this point we show how to recover,for a single sensor, set of surfaces,and reference illuminant,the set of von Kries illuminants:all lights for which von Kries adaptation is a perfect model of illumination change.To help us in this task we use a linear model of surface reflectance, but importantly,we use a local model:that is,a model derived by examining reflectance only in the region to which the sensor of interest is sensitive.Adopting such a model and treating sensors independently of one another we show that our new theory accurately predicts the good practical performance of the von Kries model