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Single-stranded positive-sense RNA viruses commonly rearrange host cell organelle membranes into neo-organelles which are involved in virus replication and assembly. These organelles serve to concentrate viral and host factors as well as to conceal viral RNA replication activities from host cell surveillance. To date, many virus-induced membrane rearrangements have been studied by targeted electron tomographic (ET) imaging of specific viral structures at timepoints of known interest. However, the broad cellular context within which these membrane modifications occur and how they change over time are not well understood. A question spanning many virus families is the morphological mechanism of formation of membrane rearrangements. Additionally, it is largely unknown how the membrane modifications affect the morphology of the organelle of origin. In this study, we address specific questions about virus-derived organelles induced by two positive-sense RNA viruses: the coronavirus mouse hepatitis virus (MHV) and the alphavirus Venezuelan equine encephalitis virus (VEEV). Utilizing serial sectioning and montage imaging for ET, volumes representing approximately 10% of virus-infected cells were imaged and detailed organelle analysis was performed. Using MHV-infected cells, we demonstrate that coronavirus-induced double-membrane vesicles (DMVs) are formed by budding from the endoplasmic reticulum (ER) and are trafficked to lysosomes for degradation. The ER remains largely morphologically normal early in infection despite the presence of hundreds of DMVs; however, late in infection, virus envelopment in the ER lumen leads to loss of cisternal morphology. For the alphavirus VEEV, we analyze the structure and origin of virus-derived cytopathic vacuoles II (CPVII). We identify four distinct morphological forms of CPVII and provide evidence that all four forms are derived from the Golgi apparatus. Additionally, a protocol is outlined for a newly-developed method for improved cell ultrastructure during genetically-encoded peroxidase tagging of membrane-proteins. This method is also amenable to ET. Overall, this work provides morphological cellular context for virus-induced membrane rearrangements from two families of positive-sense RNA viruses. Analysis of virus-host cell interactions from this large-scale ultrastructural perspective has the potential to lead to new approaches and strategies to combat current and future viral diseases.<br>

BACKGROUND: Netarsudil, a Rho kinase and norepinephrine transport (NET) inhibitor, is a new FDA approved drug used for decreasing raised intraocular pressure (IOP) in ocular hypertensive and primary open-angle glaucoma (POAG) patients. Previous studies reported that netarsudil increased outflow facility and lowered IOP by increasing active outflow areas around the circumference of the eye and dilating the episcleral veins (ESV; Kiel and Kopczynski, 2015; Ren et al., 2016). However, the mechanisms by which netarsudil increases outflow facility have not yet been fully elucidated. Moreover, the effects of netarsudil on the inner wall (IW) endothelium I-pores and B-pores of the Schlemm’s canal (SC) have also not been investigated yet. AIM: The goal was to determine if netarsudil-treatment increased the effective filtration areas (EFA) by increasing pore density in both high- and non-flow type areas, compared to untreated control eyes. METHODS: In this study, the effects of netarsudil on the pore densities on IW of SC were investigated by serial block-face scanning electron microscopy (SBF-SEM). Two pairs of eyes were perfused with green fluorescent tracers in order to determine the outflow pattern prior to treatment. Then, one eye of each pair was perfused with netarsudil, while the fellow eye of each pair was perfused with vehicle solution. All eyes were then perfused with red fluorescent tracers in order to determine the outflow pattern once they were treated with netarsudil. Both pairs of eyes were perfused and fixed at 15 mmHg. Global imaging was performed for all eyes to visualize high- and non- flow areas in the trabecular meshwork (TM) and ESV’s. A SBF-SEM was used to image eight wedges of tissue including the IW of SC and TM (high- and non-flow areas from four eyes) for a total of 16,378 images. The study analyzed the percentage of pore-types (GV-associated I-pores, Non-GV associated I-pores, B-pores), the median pore spans, the GV-associated I-pore locations, and the pore densities (per IW nuclei and IW area) between the equivalent control and netarsudil-treated flow areas. RESULTS: In global images, an increase in high-flow areas were observed in netarsudil-treated eyes due to recruitment from low-flow and non-flow areas. A greater percentage of GV-associated I-pores, B-pores, and total pores were found in high-flow in contrast to non-flow areas in both control and netarsudil-treated eyes (all P ≤ 0.05). However, the percentage of GV-associated I-pores in non-flow areas were significantly greater in treated compared to control eyes (P ≤ 0.05). Qualitative observations from two pairs of eyes showed a trend of greater I-pore, B-pore, and total pore density/per IW nucleus and density/per IW surface area in high-flow in contrast to non-flow areas for both treated and control eyes. No difference in I-pore, B-pore, and total pore density/per IW nucleus and density /per IW surface area were observed in equivalent flow-type areas when comparing control and netarsudil-treated eyes. In addition, there was a significant greater percentage of I-pores located on the side of GVs than the top of GVs in all cases (P ≤ 0.05). CONCLUSIONS: Netarsudil increased high-flow areas. A greater pore density was found in high-flow in contrast to non-flow areas. Netarsudil also significantly increased the proportion of GV-associated I-pores in non-flow areas when compared to control eyes. Our results suggests that one mechanism of netarsudil increasing outflow facility is acting through recruiting the high-flow areas around the circumference of the eye, which is associated with higher pore density and increasing the proportion of GV-associated I-pores in non-flow areas.

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Salmonellen spp. sind invasive, intrazelluläre Pathogene, die in einem membranumhüllten Kompartiment innerhalb der infizierten Wirtszelle überleben. Wie auch andere intrazelluläre Pathogene repliziert Salmonella in dieser intrazellulären Nische, obwohl es anscheinend von sowohl extra- als auch intrazellulären Nährstoffquellen isoliert ist. Wir zeigen hier, dass intrazelluläre Salmonella den Proteinabbau des Wirts ausnutzen, um Aminosäuren in Form von Peptiden zu erhalten. Dieser spezielle, auch als Chaperon-vermittelte Autophagie bekannte, Abbauweg spielt eine Rolle im Transport zytosolischer Proteine zum Abbau im Lysosom. Ein Salmonellenmutant, der nur in Anwesenheit von Peptiden im Medium als Aminosäurenquelle wächst, wies intrazellulär eine Wachstumsrate auf, die der des Wildtyps ähnlich war. Dies deutet darauf hin, dass Peptide intrazellulär für Salmonella zugänglich sind. Wir fanden heraus, dass die Salmonella-enthaltende Vakuole (SCV, Salmonella containing vacuole) die Wirtproteine LAMP-2A und Hsc73, Kernkomponenten von CMA, anzieht, jedoch nicht lysosomale Proteine wie LAMP-2B und LIMP-2. Im Gegensatz zum Salmonellawildtyp zeigte der peptidabhängige Mutantentstamm stark verringertes Wachstum, wenn die Wirtszellen mit CMA-Inhibitoren behandelt wurde. Diese Ergebnisse zeigen einen neuen Mechanismus auf, durch den ein intrazelluläres Pathogen vom membranumhüllten Kompartiment aus Zugriff auf Cytosol der Wirtzelle zur Beschaffung von Nährstoffen hat. Wir schlagen vor, dass diese Ergebnisse eine Erklärung für die Rückfälle von persistenten Salmonellainfektionen liefern können. Des Weitern schlagen wir diesen Mechanismus als moegliches Ziel antibakterieller Therapeutika zur Bekämpfung intrazellulärer Pathogene vor. / Salmonella spp. are invasive, intracellular pathogens which survive and proliferate within a membrane-bound compartment inside infected host cells. Like other intracellular pathogens, Salmonella replicates within this intracellular niche, despite its apparent isolation from both extra- and intracellular sources of nutrients. Here, we show that intracellular Salmonella acquire amino acids in the form of peptides by co-opting the host protein degradation pathway known as chaperone-mediated autophagy (CMA) involved in the transport of cytosolic proteins to the lysosome for degradation. A mutant of Salmonella strictly dependent upon peptides in growth media as a source of amino acids, showed intracellular growth similar to the wild-type strain in host cells, indicating intracellular access to peptides. We found that the Salmonella-containing vacuole (SCV) acquires the host cell proteins LAMP-2A and Hsc73, key components of CMA, but excludes lysosomal proteins such as LAMP-2B and LIMP-2. In contrast to wild-type Salmonella, the peptide-dependent mutant strain showed a severe reduction in growth when host cells were treated with inhibitors of CMA.. These results reveal a novel means whereby an intracellular pathogen can access the host cell cytosol to acquire nutrients from within its membrane-bound compartment. We suggest these results may provide an explanation for relapse infections resulting from persistent Salmonella infections, and suggest a possible means of targeting antibacterials against intracellular pathogens.

The genus Salmonella includes facultative intracellular pathogens. Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever in humans killing about 2,00,000 people globally every year. Salmonella enterica serovars Typhimurium (S. Typhimurium) and Enteritidis (S. Enteritidis) cause food poisoning in humans. Salmonellae also cause disease in animals of economic importance like poultry and cattle. Treatment of diseases caused by these notorious pathogens is becoming more and more difficult because of the emergence of drug resistant strains. Thus, it is vital to understand the virulence mechanisms of Salmonella which can lead us to potential drug targets and also help us design effective vaccines. Salmonella has evolved many strategies to enter the host, to evade intracellular and extracellular antimicrobial activities of the host and to extract nutrition in the stringent and hostile environment of the host. These strategies have enabled Salmonella to survive and multiply in the host making it a successful pathogen. Present study deals with four such survival strategies of Salmonella. S. Typhimurium causes a systemic disease in mice that is similar to typhoid fever caused by serovar Typhi in humans. This serves as a good model system to study and understand the pathogenesis of Salmonellae. This model system has been used throughout this study. In the present thesis attempts have been made to identify some novel survival strategies of Salmonella. The thesis is divided into five chapters. Chapter 1 gives an introduction into the basic biology of these notorious pathogens. The diseases caused by Salmonellae are introduced in this chapter. Typhoid fever is discussed in detail covering its epidemiology, clinical features, diagnosis, treatment and prevention. Next section covers the virulence determinants of Salmonella. In this section, Salmonella pathogenicity islands are discussed in detail. This chapter concludes with an overview of molecular pathogenesis of Salmonella covering its invasion strategy and its dangerous life inside the host cell. Salmonella stays and multiplies inside a specialized endosomal compartment of the host cell known as Salmonella-containing vacuole (SCV). It is believed that Salmonella multiplies inside SCV resulting in single big vacuole containing multiple bacteria. The results of Chapter 2 challenge this notion. Using transmission electron microscopy and confocal laser scanning microscopy we show that SCV also divides along with the division of Salmonella resulting in multiple SCVs containing single bacterium per vacuole. We also show that this division is mediated by the molecular motor dynein. This chapter concludes with a discussion on the advantages of SCV division with respect to Salmonella. Successful intracellular pathogens must have some strategy either to avoid lysosomal fusion or to endure the toxic molecules of lysosomes. In case of Salmonella, it is well accepted that SCV-lysosome fusion is blocked. However, the exact mechanism of this process is still unclear. The results of Chapter 3 enhance our understanding of this issue. This chapter explores an interesting possibility of Salmonella reducing the lysosomal number and thereby reducing the chances of SCV-lysosome fusion. Using flowcytometry and confocal laser scanning microscopy, we show that Salmonella decreases the number of acidic lysosomes in murine macrophages. Thus, our results suggest that there is an imbalance in the ratio of vacuoles to acidic lysosomes which decreases the probability of SCV-lysosome fusion thereby helping Salmonella avoid lysosomes. Multicellular organisms use various defense strategies to protect themselves from microbial infections; production of antimicrobial peptides (AMPs) is one of them. Being cationic in nature, AMPs interact and cause pores in the bacterial membrane eventually killing the bacteria. Pathogenic micro-organisms like Salmonella have evolved many strategies to counteract the AMPs they encounter upon their entry into the host systems. S Typhimurium genome has a gene cluster consisting of yejA, yejB, yejE and yejF genes which encode a putative ABC transporter. Chapter 4 deals with the detailed characterization of these genes. Our study shows that these genes constitute an operon. We have deleted the yejF gene which encodes the ATPase component of this putative ABC transporter. The ΔyejF strain showed increased sensitivity to AMPs like protamine, melittin, polymyxin B and human defensins and was compromised to proliferate inside activated macrophages and epithelial cells. In murine typhoid model, the ΔyejF strain displayed decreased virulence when infected intragastrically. These findings suggest that the putative transporter encoded by the yejABEF operon is involved in counteracting AMPs and contributes to the virulence of Salmonella. An important biochemical property of Salmonella that distinguishes it from the closely related E. coli is its inability to ferment lactose. In E. coli, lactose fermentation is carried out by the products of lac operon which is regulated by a repressor encoded by lacI. Salmonella does not have the lac operon and lacI. It has been proposed that S.enterica has lost lac region (lacI and lacZYA) during its evolution. Chapter 5 deals with the evolutionary and physiological significance behind the loss of lac region by S.enterica. We show that expression of LacI in S. enterica suppresses its virulence by interfering with the expression of SPI-2 virulence genes. We also observed that the genome of S. bongori which does not have the virulence genes of SPI-2 has a homologue of LacI. Our results suggest that presence of lacI has probably hindered the acquisition of virulence genes of SPI-2 in S. bongori, whereas absence of lacI has facilitated the same in S. enterica making it a successful systemic pathogen. Thus, lacI has played a remarkable role in the evolution of Salmonella virulence. Brief summary of four studies that are not directly related to survival strategies of Salmonella are included in Appendix. First two studies analyze molecular evolution of SPIs to understand the mechanism of host specificity in Salmonella and the last two studies explore the signaling of lipopolysaccharide (LPS) derived from Salmonella.

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Vitellogenesis in two caryophyllidean cestodes Caryophyllaeus laticeps and Khawia sinensis, parasitizing cyprinid fishes, were examined using light(LM)and transmission electron microscopy(TEM)and cytochemical staining for glycogen. Mature vitelline folicles consist of vitelline cells at various stages of development and an interstitial tissue. Maturing and mature vitellocytes contain vitelline material in the form of single small shell globules, which fuse and give rise to the large shell globule clusters.Glycogen was present in the cytoplasm and in the nucleus of the mature vitellocytes. Small lipid droplets were found in the cytoplasm of C. laticeps. "Lamellar granules" were observed in the cytoplasm of the mature vitellocytes in K. sinensis.