STRUCTURED CORRELATION DETECTION WITH APPLICATION TO COLOCALIZATION ANALYSIS IN DUAL-CHANNEL FLUORESCENCE MICROSCOPIC IMAGING.

Current workflows for colocalization analysis in fluorescence microscopic imaging introduce significant bias in terms of the user's choice of region of interest (ROI). In this work, we introduce an automatic, unbiased structured detection method for correlated region detection between two random processes observed on a common domain. We argue that although intuitive, using the maximum log-likelihood statistic directly suffers from potential bias and substantially reduced power. Therefore, we introduce a simple size-based normalization to overcome this problem. We show that scanning using the proposed statistic leads to optimal correlated region detection over a large collection of structured correlation detection problems.

Mapping of Shigella flexneri's tissue distribution and type III secretion apparatus activity during infection of the large intestine of guinea pigs.

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo.

Shigella-mediated oxygen depletion is essential for intestinal mucosa colonization.

Pathogenic enterobacteria face various oxygen (O2) levels during intestinal colonization from the O2-deprived lumen to oxygenated tissues. Using Shigella flexneri as a model, we have previously demonstrated that epithelium invasion is promoted by O2 in a type III secretion system-dependent manner. However, subsequent pathogen adaptation to tissue oxygenation modulation remained unknown. Assessing single-cell distribution, together with tissue oxygenation, we demonstrate here that the colonic mucosa O2 is actively depleted by S. flexneri aerobic respiration-and not host neutrophils-during infection, leading to the formation of hypoxic foci of infection. This process is promoted by type III secretion system inactivation in infected tissues, favouring colonizers over explorers. We identify the molecular mechanisms supporting infectious hypoxia induction, and demonstrate here how enteropathogens optimize their colonization capacity in relation to their ability to manipulate tissue oxygenation during infection.

Scientific Community Image Forum: A discussion forum for scientific image software.

Forums and email lists play a major role in assisting scientists in using software. Previously, each open-source bioimaging software package had its own distinct forum or email list. Although each provided access to experts from various software teams, this fragmentation resulted in many scientists not knowing where to begin with their projects. Thus, the scientific imaging community lacked a central platform where solutions could be discussed in an open, software-independent manner. In response, we introduce the Scientific Community Image Forum, where users can pose software-related questions about digital image analysis, acquisition, and data management.

Spatially Adaptive Colocalization Analysis in Dual-Color Fluorescence Microscopy.

Colocalization analysis aims to study complex spatial associations between bio-molecules via optical imaging techniques. However, existing colocalization analysis workflows only assess an average degree of colocalization within a certain region of interest and ignore the unique and valuable spatial information offered by microscopy. In the current work, we introduce a new framework for colocalization analysis that allows us to quantify colocalization levels at each individual location and automatically identify pixels or regions where colocalization occurs. The framework, referred to as spatially adaptive colocalization analysis (SACA), integrates a pixel-wise local kernel model for colocalization quantification and a multi-scale adaptive propagation-separation strategy for utilizing spatial information to detect colocalization in a spatially adaptive fashion. Applications to simulated and real biological datasets demonstrate the practical merits of SACA in what we hope to be an easily applicable and robust colocalization analysis method. In addition, theoretical properties of SACA are investigated to provide rigorous statistical justification.

Disentangling Host-Microbiota Regulation of Lipid Secretion by Enterocytes: Insights from Commensals Lactobacillus paracasei and Escherichia coli.

The gut microbiota contributes to nutrients absorption and metabolism by enterocytes, but the molecular mechanisms involved remain poorly understood, and most conclusions are inferred from studies comparing germfree and conventional animals colonized with diverse bacterial species. We selected two model commensal microorganisms, Escherichia coli and Lactobacillus paracasei, to assess the role of the small-intestinal microbiota in modulating lipid absorption and metabolism by the epithelium. Using an integrated approach encompassing cellular and murine models and combining metabolic parameters measurement, lipid droplet imaging, and gene expression analysis, we demonstrated that under homeostatic conditions, L. paracasei promotes fat storage in enterocytes, whereas E. coli enhances lipid catabolism and reduces chylomicron circulating levels. The Akt/mammalian target of sirolimus (mTOR) pathway is inhibited by both bacterial species in vitro, indicating that several regulatory pathways are involved in the distinct intracellular lipid outcomes associated with each bacterial species. Moreover, soluble bacterial factors partially reproduce the effects observed with live microorganisms. However, reduction of chylomicron circulating levels in E. coli-colonized animals is lost under high-fat-diet conditions, whereas it is potentiated by L. paracasei colonization accompanied by resistance to hypercholesterolemia and excess body weight gain.IMPORTANCE The specific contribution of each bacterial species within a complex microbiota to the regulation of host lipid metabolism remains largely unknown. Using two model commensal microorganisms, L. paracasei and E. coli, we demonstrated that both bacterial species impacted host lipid metabolism in a diet-dependent manner and, notably, that L. paracasei-colonized mice but not E. coli-colonized mice resisted high-fat-diet-induced body weight gain. In addition, we set up cellular models of fatty acid absorption and secretion by enterocytes cocultured with bacteria and showed that, in vitro, both L. paracasei and E. coli inhibited lipid secretion, through increased intracellular fat storage and enhanced lipid catabolism, respectively.

MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases.

Automated and Robust Quantification of Colocalization in Dual-Color Fluorescence Microscopy: A Nonparametric Statistical Approach.

Colocalization is a powerful tool to study the interactions between fluorescently labeled molecules in biological fluorescence microscopy. However, existing techniques for colocalization analysis have not undergone continued development especially in regards to robust statistical support. In this paper, we examine two of the most popular quantification techniques for colocalization and argue that they could be improved upon using ideas from nonparametric statistics and scan statistics. In particular, we propose a new colocalization metric that is robust, easily implementable, and optimal in a rigorous statistical testing framework. Application to several benchmark data sets, as well as biological examples, further demonstrates the usefulness of the proposed technique.

ImageJ2: ImageJ for the next generation of scientific image data.

BACKGROUND: ImageJ is an image analysis program extensively used in the biological sciences and beyond. Due to its ease of use, recordable macro language, and extensible plug-in architecture, ImageJ enjoys contributions from non-programmers, amateur programmers, and professional developers alike. Enabling such a diversity of contributors has resulted in a large community that spans the biological and physical sciences. However, a rapidly growing user base, diverging plugin suites, and technical limitations have revealed a clear need for a concerted software engineering effort to support emerging imaging paradigms, to ensure the software's ability to handle the requirements of modern science. RESULTS: We rewrote the entire ImageJ codebase, engineering a redesigned plugin mechanism intended to facilitate extensibility at every level, with the goal of creating a more powerful tool that continues to serve the existing community while addressing a wider range of scientific requirements. This next-generation ImageJ, called "ImageJ2" in places where the distinction matters, provides a host of new functionality. It separates concerns, fully decoupling the data model from the user interface. It emphasizes integration with external applications to maximize interoperability. Its robust new plugin framework allows everything from image formats, to scripting languages, to visualization to be extended by the community. The redesigned data model supports arbitrarily large, N-dimensional datasets, which are increasingly common in modern image acquisition. Despite the scope of these changes, backwards compatibility is maintained such that this new functionality can be seamlessly integrated with the classic ImageJ interface, allowing users and developers to migrate to these new methods at their own pace. CONCLUSIONS: Scientific imaging benefits from open-source programs that advance new method development and deployment to a diverse audience. ImageJ has continuously evolved with this idea in mind; however, new and emerging scientific requirements have posed corresponding challenges for ImageJ's development. The described improvements provide a framework engineered for flexibility, intended to support these requirements as well as accommodate future needs. Future efforts will focus on implementing new algorithms in this framework and expanding collaborations with other popular scientific software suites.

Quantitating the cell: turning images into numbers with ImageJ.

Modern biological research particularly in the fields of developmental and cell biology has been transformed by the rapid evolution of the light microscope. The light microscope, long a mainstay of the experimental biologist, is now used for a wide array of biological experimental scenarios and sample types. Much of the great developments in advanced biological imaging have been driven by the digital imaging revolution with powerful processors and algorithms. In particular, this combination of advanced imaging and computational analysis has resulted in the drive of the modern biologist to not only visually inspect dynamic phenomena, but to quantify the involved processes. This need to quantitate images has become a major thrust within the bioimaging community and requires extensible and accessible image processing routines with corresponding intuitive software packages. Novel algorithms both made specifically for light microscopy or adapted from other fields, such as astronomy, are available to biologists, but often in a form that is inaccessible for a number of reasons ranging from data input issues, usability and training concerns, and accessibility and output limitations. The biological community has responded to this need by developing open source software packages that are freely available and provide access to image processing routines. One of the most prominent is the open-source image package ImageJ. In this review, we give an overview of prominent imaging processing approaches in ImageJ that we think are of particular interest for biological imaging and that illustrate the functionality of ImageJ and other open source image analysis software. WIREs Dev Biol 2017, 6:e260. doi: 10.1002/wdev.260 For further resources related to this article, please visit the WIREs website.

The infectious hypoxia: occurrence and causes during Shigella infection.

Hypoxia is defined as a tissue oxygenation status below physiological needs. During Shigella infection, an infectious hypoxia is induced within foci of infection. In this review, we discuss how Shigella physiology and virulence are modulated and how the main recruited immune cells, the neutrophils, adapt to this environment.

Bioimage analysis of Shigella infection reveals targeting of colonic crypts.

Few studies within the pathogenic field have used advanced imaging and analytical tools to quantitatively measure pathogenicity in vivo. In this work, we present a novel approach for the investigation of host-pathogen processes based on medium-throughput 3D fluorescence imaging. The guinea pig model for Shigella flexneri invasion of the colonic mucosa was used to monitor the infectious process over time with GFP-expressing S. flexneri. A precise quantitative imaging protocol was devised to follow individual S. flexneri in a large tissue volume. An extensive dataset of confocal images was obtained and processed to extract specific quantitative information regarding the progression of S. flexneri infection in an unbiased and exhaustive manner. Specific parameters included the analysis of S. flexneri positions relative to the epithelial surface, S. flexneri density within the tissue, and volume of tissue destruction. In particular, at early time points, there was a clear association of S. flexneri with crypts, key morphological features of the colonic mucosa. Numerical simulations based on random bacterial entry confirmed the bias of experimentally measured S. flexneri for early crypt targeting. The application of a correlative light and electron microscopy technique adapted for thick tissue samples further confirmed the location of S. flexneri within colonocytes at the mouth of crypts. This quantitative imaging approach is a novel means to examine host-pathogen systems in a tailored and robust manner, inclusive of the infectious agent.

Apical invasion of intestinal epithelial cells by Salmonella typhimurium requires villin to remodel the brush border actin cytoskeleton.

Salmonella invasion of intestinal epithelial cells requires extensive, though transient, actin modifications at the site of bacterial entry. The actin-modifying protein villin is present in the brush border where it participates in the constitution of microvilli and in epithelial restitution after damage through its actin-severing activity. We investigated a possible role for villin in Salmonella invasion. The absence of villin, which is normally located at the bacterial entry site, leads to a decrease in Salmonella invasion. Villin is necessary for early membrane-associated processes and for optimal ruffle assembly by balancing the steady-state level of actin. The severing activity of villin is important for Salmonella invasion in vivo. The bacterial phosphatase SptP tightly regulates villin phosphorylation, while the actin-binding effector SipA protects F-actin and counterbalances villin-severing activity. Thus, villin plays an important role in establishing the balance between actin polymerization and actin severing to facilitate the initial steps of Salmonella entry.

B lymphocytes undergo TLR2-dependent apoptosis upon Shigella infection.

Antibody-mediated immunity to Shigella, the causative agent of bacillary dysentery, requires several episodes of infection to get primed and is short-lasting, suggesting that the B cell response is functionally impaired. We show that upon ex vivo infection of human colonic tissue, invasive S. flexneri interacts with and occasionally invades B lymphocytes. The induction of a type three secretion apparatus (T3SA)-dependent B cell death is observed in the human CL-01 B cell line in vitro, as well as in mouse B lymphocytes in vivo. In addition to cell death occurring in Shigella-invaded CL-01 B lymphocytes, we provide evidence that the T3SA needle tip protein IpaD can induce cell death in noninvaded cells. IpaD binds to and induces B cell apoptosis via TLR2, a signaling receptor thus far considered to result in activation of B lymphocytes. The presence of bacterial co-signals is required to sensitize B cells to apoptosis and to up-regulate tlr2, thus enhancing IpaD binding. Apoptotic B lymphocytes in contact with Shigella-IpaD are detected in rectal biopsies of infected individuals. This study therefore adds direct B lymphocyte targeting to the diversity of mechanisms used by Shigella to dampen the host immune response.

Enterohepatic bacterial infections dysregulate the FGF15-FGFR4 endocrine axis.

BACKGROUND: Enterohepatic bacterial infections have the potential to affect multiple physiological processes of the body. Fibroblast growth factor 15/19 (FGF15 in mice, FGF19 in humans) is a hormone that functions as a central regulator of glucose, lipid and bile acid metabolism. FGF15/19 is produced in the intestine and exert its actions on the liver by signaling through the FGFR4-ßKlotho receptor complex. Here, we examined the in vivo effects of enterohepatic bacterial infection over the FGF15 endocrine axis. RESULTS: Infection triggered significant reductions in the intestinal expression of Fgf15 and its hepatic receptor components (Fgfr4 and Klb (ßKlotho)). Infection also resulted in alterations of the expression pattern of genes involved in hepatobiliary function, marked reduction in gallbladder bile volumes and accumulation of hepatic cholesterol and triglycerides. The decrease in ileal Fgf15 expression was associated with liver bacterial colonization and hepatobiliary pathophysiology rather than with direct intestinal bacterial pathogenesis. CONCLUSIONS: Bacterial pathogens of the enterohepatic system can disturb the homeostasis of the FGF15/19-FGFR4 endocrine axis. These results open up a possible link between FGF15/19-FGFR4 disruptions and the metabolic and nutritional disorders observed in infectious diseases.

Shigella impairs T lymphocyte dynamics in vivo.

The Gram-negative enteroinvasive bacterium Shigella flexneri is responsible for the endemic form of bacillary dysentery, an acute rectocolitis in humans. S. flexneri uses a type III secretion system to inject effector proteins into host cells, thus diverting cellular functions to its own benefit. Protective immunity to reinfection requires several rounds of infection to be elicited and is short-lasting, suggesting that S. flexneri interferes with the priming of specific immunity. Considering the key role played by T-lymphocyte trafficking in priming of adaptive immunity, we investigated the impact of S. flexneri on T-cell dynamics in vivo. By using two-photon microscopy to visualize bacterium-T-cell cross-talks in the lymph nodes, where the adaptive immunity is initiated, we provide evidence that S. flexneri, via its type III secretion system, impairs the migration pattern of CD4(+) T cells independently of cognate recognition of bacterial antigens. We show that bacterial invasion of CD4(+) T lymphocytes occurs in vivo, and results in cell migration arrest. In the absence of invasion, CD4(+) T-cell migration parameters are also dramatically altered. Signals resulting from S. flexneri interactions with subcapsular sinus macrophages and dendritic cells, and recruitment of polymorphonuclear cells are likely to contribute to this phenomenon. These findings indicate that S. flexneri targets T lymphocytes in vivo and highlight the role of type III effector secretion in modulating host adaptive immune responses.

Oxysterol-binding protein (OSBP) enhances replication of intracellular Salmonella and binds the Salmonella SPI-2 effector SseL via its N-terminus.

Effectors translocated into the host cell by Salmonella enterica serovar Typhimurium are critical for bacterial virulence. For many effectors, the mechanisms of their interactions with host pathways are not yet understood. We have recently found an interaction between the SPI-2 effector SseL and oxysterol-binding protein (OSBP). We show here that SseL binds the N-terminus of OSBP and that S. Typhimurium infection results in redistribution of OSBP. We furthermore demonstrate that OSBP is required for efficient replication of intracellular S. Typhimurium. This suggests that S. Typhimurium hijacks OSBP-dependent pathways to benefit its intracellular life-style, possibly by SseL- and OSBP-mediated manipulation of host lipid metabolism.

The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets.

To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

Metabolomics reveals phospholipids as important nutrient sources during Salmonella growth in bile in vitro and in vivo.

During the colonization of hosts, bacterial pathogens are presented with many challenges that must be overcome for colonization to occur successfully. This requires the bacterial sensing of the surroundings and adaptation to the conditions encountered. One of the major impediments to the pathogen colonization of the mammalian gastrointestinal tract is the antibacterial action of bile. Salmonella enterica serovar Typhimurium has specific mechanisms involved in resistance to bile. Additionally, Salmonella can successfully multiply in bile, using it as a source of nutrients. This accomplishment is highly relevant to pathogenesis, as Salmonella colonizes the gallbladder of hosts, where it can be carried asymptomatically and promote further host spread and transmission. To gain insights into the mechanisms used by Salmonella to grow in bile, we studied the changes elicited by Salmonella in the chemical composition of bile during growth in vitro and in vivo through a metabolomics approach. Our data suggest that phospholipids are an important source of carbon and energy for Salmonella during growth in the laboratory as well as during gallbladder infections of mice. Further studies in this area will generate a better understanding of how Salmonella exploits this generally hostile environment for its own benefit.

A comprehensive study of the contribution of Salmonella enterica serovar Typhimurium SPI2 effectors to bacterial colonization, survival, and replication in typhoid fever, macrophage, and epithelial cell infection models.

Salmonella enterica serovars are Gram-negative bacterial pathogens responsible for human diseases including gastroenteritis and typhoid fever. After ingestion, Salmonella cross the intestinal epithelial barrier, where they are phagocytosed by macrophages and dendritic cells, which then enables their spread to systemic sites during cases of typhoid fever. Salmonella use two type 3 secretion systems encoded by Salmonella pathogenicity islands (SPI) 1 and 2 to inject virulence proteins into host cells to modify cellular functions. SPI1 is involved in host cell invasion and inflammation, whereas SPI2 is required for intracellular survival and replication within phagocytes, and systemic spread. In this study the contribution of nearly all known SPI2 effectors was examined in an in vivo model of murine typhoid fever and cell culture models of macrophage and epithelial cell infection. Unmarked, in-frame deletions of SPI2 effectors were engineered in S. enterica serovar Typhimurium and the ability of the 16 different mutants to colonize and replicate was examined. In the typhoid model, we found that ΔspvB and ΔspiC mutants were attenuated for colonization of intestinal and systemic sites, while the ΔsseF mutant was attenuated in systemic organs. In epithelial cells, all mutants replicated to the same extent as the wild-type. In macrophages, ΔspiC, ΔsteC, ΔspvB, ΔssseK1/K2/K3, ΔsifA, and ΔsifB strains replicated poorly in comparison to wild-type Salmonella. This study provides a thorough screen of the majority of the known SPI2 effectors evaluated under the same conditions in various models of infection, providing a foundation for comparative examination of the roles and interactions of these effectors.