Subversion of membrane transport pathways by vacuolar pathogens

Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase RAS and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA(2) are activated and are important for the onset of inflammatory responses. In this study we tested if activation of ERK and cPLA(2) occurred as a result of RAS signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. We provide genetic and pharmacological evidence that during infection RAS, ERK, and, to a lesser extent, cPLA(2) activation are uncoupled, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA(2) signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells, inhibition of ERK and cPLA(2) played a significant protective role against C. trachomatis. We determined that cPLA(2)-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of beta interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Furthermore, the MAPK p38 was primarily responsible for cPLA(2) activation in Chlamydia-infected cells and IRG expression. Overall, these findings define a previously unrecognized role for cPLA(2) in the induction of cell autonomous cellular immunity to Chlamydia and highlight the many non-canonical signaling pathways engaged during infection.

The phylogenetic tree segregates into three major groups, which probably descended from a common ancestor: the bacteria, the archaea, and the eucarya (1–3). While bacteria and archaea lack a true nucleus and intracellular organelles, eucarya possess these. The origin of the ancestor remains elusive, but it emerged about 4 billion years ago. The bacteria probably started life 3.5 billion years ago and eukaryotes emerged 1.75 billion years thereafter. Sometime in between archaea appeared. Eukaryotes began as unicellular organisms but soon also developed multicellular forms. Bacteria and archaea had sufficient time to exploit all niches of the abiotic environment, ranging from ice cold glacial lakes to fiery geysers – from the highest mountain peaks to the deepest sea beds. They also quickly developed forms of mutualism by forming colonies composed of mixed populations. With the evolution of eucarya, new environments could be exploited. Today prokaryotes are the most prevalent form of life on earth, making up the majority of our biomass. It has been estimated that more than 10 prokaryotic organisms live on our globe. Thus, archaea and bacteria are far more prevalent than eucarya. Even human beings comprise more prokaryotic cells than mammalian cells, which range on the order of 10 human cells and 10 prokaryotes mostly concentrated in our intestinal system (4–7).

The CBL-CIPK Calcium Signaling Network: Unified Paradigm from 20 Years of Discoveries.

Obligate intracellular pathogens occupy one of two niches - free in the host cell cytoplasm or confined in a membrane-bound vacuole. Pathogens occupying membrane-bound vacuoles are sequestered from the innate immune system and have an extra layer of protection from antimicrobial drugs. However, this lifestyle presents several challenges. First, the bacteria must obtain membrane or membrane components to support vacuole expansion and provide space for the increasing bacteria numbers during the log phase of replication. Second, the vacuole microenvironment must be suitable for the unique metabolic needs of the pathogen. Third, as most obligate intracellular bacterial pathogens have undergone genomic reduction and are not capable of full metabolic independence, the bacteria must have mechanisms to obtain essential nutrients and resources from the host cell. Finally, because they are separated from the host cell by the vacuole membrane, the bacteria must possess mechanisms to manipulate the host cell, typically through a specialized secretion system which crosses the vacuole membrane. While there are common themes, each bacterial pathogen utilizes unique approach to establishing and maintaining their intracellular niches. In this review, we focus on the vacuole-bound intracellular niches of Anaplasma phagocytophilum, Ehrlichia chaffeensis, Chlamydia trachomatis, and Coxiella burnetii.

Specific D-glucose transport in plasma membranes prepared from control and insulin-treated rat adipocytes was measured using a recently developed dual isotope ([3H]-D-glucose and [14C]-L-glucose), rapid filtration assay which allowed measurements of initial rates at 1 s and 37 degrees. Plasma membranes from insulin-treated adipocytes showed an increase in glucose transport compared with control cells. Saturation kinetic data revealed that the plasma membranes from insulin-treated and control cells had the same Km (26 mM) for glucose transport, whereas insulin treatment increased the Vmax from 4433 pmol/mg protein/s to 9465 pmol/mg proteins/s. Arrhenius plots showed no difference in the energy of activation between control and insulin-stimulated glucose transport states. The optimum pH of both control and insulin-stimulated glucose transport was 7.4. Lower or higher pHs progressively decreased both control and insulin-stimulated glucose transport proportionately. Calcium in the transport assay media did not affect basal or insulin-stimulated glucose transport. However, omission of calcium from the adipocyte incubation media significantly lowered the insulin stimulation by 24% while basal levels were not significantly affected. Insulin specifically bound to the plasma membrane was carried through the fractionation procedure, but removal of this insulin did not alter the stimulated glucose transport. Glucose transport by plasma membranes from control or insulin-treated adipocytes was equally (percentage) inhibited by N-ethylmaleimide, dithiothreitol, reduced glutathione, or cytochalasin B. No inhibition of control or insulin-stimulated transport was seen with cytochalasin D or oxidized glutathione. The data presented are consistent with insulin causing the formation of new transport sites similar to the existing basal sites.

Regulation of the microtubule cytoskeleton is of pivotal importance for neuronal development and function. One such regulatory mechanism centers on microtubule plus-end tracking proteins (+TIPs): structurally and functionally diverse regulatory factors, which can form complex macromolecular assemblies at the growing microtubule plus-ends. +TIPs modulate important properties of microtubules including their dynamics and their ability to control cell polarity, membrane transport and signaling. Several neurodevelopmental and neurodegenerative diseases are associated with mutations in +TIPs or with misregulation of these proteins. In this review, we focus on the role and regulation of +TIPs in neuronal development and associated disorders.

The invasion of unicellular organisms into other organisms’ cells is widespread among bacteria, protozoa, and fungi. This phenomenon is most often manifested as intracellular parasitism, resulting in serious diseases in humans, animals, and plants. Intracellular pathogen species include about 20 bacterial taxa that can cause serious diseases in humans and animals, such as tuberculosis, leprosy, typhus, salmonellosis, brucellosis, trachoma, dysentery, and other. This review considers in detail the survival strategies of various intracellular bacterial pathogens: their mechanisms of interaction with the host cell membrane, techniques of intrusion into the host cell, survival strategies within the host cell, and mechanisms to avoid host immune responses. We also discuss the strategies of latent persistence of certain bacterial intracellular pathogens, which permit them to survive in the host organism in an inactive form for a long time under conditions of high immune pressure and become rapidly reactivated when the host immune-response levels are reduced.

### Contents #### News [The Roots of Plant-Microbe Collaborations][1] #### Reviews How Viruses Enter Animal Cells A. E. Smith and A. Helenius [Bacterial Invasion: The Paradigms of Enteroinvasive Pathogens][2] P. Cossart and P. J. Sansonetti Intracellular Parasite Invasion Strategies L. D. Sibley Ancient Invasions: From Endosymbionts to Organelles S. D. Dyall et al. See related Science Express by [Horn et al.][3] I n many biological systems, one organism, often a virus, bacterium, or other microbe, invades another, generally at the cellular level. The ramifications can be profound. For example, a virus infecting a single animal cell can ultimately lead to the demise of the whole animal. Other microbial parasites may also have catastrophic consequences for their hosts, as seen in the terrible toll taken by malarial parasites in the develoing world. Cellular invasions can also have more benign outcomes, as when microbial symbioses are established in the roots of legumes, allowing them to gain essential nitrogen nutrients; or in our ancient past, when bacteria took up residence in prototype eukaryotic cells and evolved into mitochondria, the cell's energy powerhouses. In this special issue, we look at a variety of these cellular invasions and examine the intracellular niches established by invading microorganisms. Smith and Helenius (p. 237) describe invasions by animal viruses, which employ multiple strategies to allow the viral nucleic acids to gain access to the host cell replication machinery and generate progeny viruses. Some bacterial pathogens can also take up residence within eukaryotic host cells. Cossart and Sansonetti (p. [242][2]) look at infections by a group of enteroinvasive bacteria, including Salmonella , Shigella, Yersinia , and Listeria , that cause certain types of food poisoning. When these microbes invade cells, they establish a privileged intracellular niche within which they can grow and divide, protected from host defense mechanisms. In Science Express, Horn et al. ([www.sciencemag.org/cgi/content/abstract/1096330][3]) present a comparative and phylogenetic genome analysis of a chlamydia-related symbiont of free-living amoebas. They suggest that ancient chlamydiae were major inventors of mechanisms used by intracellular bacterial pathogens for survival in, and exploitation of, eukaryotic host cells. Another group of invasive organisms, the apicomplexan parasites, includes Plasmodium , which causes malaria, and the organism responsible for toxoplasmosis, which can damage multiple organs in immune-compromised individuals. Sibley (p. 248) reviews how apicomplexan parasites gain entry to susceptible host cells. Dyall et al. (p. 253) describe how, way back in evolutionary history, endosymbionts developed into organelles such as mitochondria and chloroplasts, thus producing early eukaryotes. In an accompanying News story (p. [234][1]), Jean Marx looks at the molecular interactions required for the establishment of two types of intracellular symbioses that are critical for plant growth and thus for modern agriculture: In one, nitrogen-fixing rhizobial bacteria set up housekeeping in legume roots, and in the other, mycorrhizal fungi invade the roots of many plants and help them to acquire phosphate. Keeping yourself to yourself may be the done thing in polite society, but in the cut and thrust of the real life of invasive organisms, it's simply not an option. [1]: /lookup/doi/10.1126/science.304.5668.234 [2]: /lookup/doi/10.1126/science.1090124 [3]: http://www.sciencemag.org/cgi/content/abstract/1096330

A Rab-Centric Perspective of Bacterial Pathogen-Occupied Vacuoles

Flagella are sophisticated nanomachines and important virulence factors of many pathogenic bacteria. Besides the ability to swim through liquid environments, the flagellum also contributes to successful infection of the host cell by enhancing cell adhesion and invasion. The flagellum consists out of three main parts: (i) the basal body, (ii) a flexible hook, and (iii) a long helical filament. Due to its purposes in distinct infection phases, flagellar gene expression, assembly, and functionality has to be regulated tightly in response to environmental cues. In this thesis, new motility regulators have been identified and mechanisms of flagella modification have been characterised according to their contribution to motility and pathogenesis of Salmonella. In the first chapter, five genes (rflP, yjcC, STM1267, STM3363, and rfaG) have been shown to influence swimming and/or swarming motility via alterations of flagellar gene expression or assembly. In the second chapter, a recently described transcriptional activator of flagellar gene expression, HilD, was characterised regarding its contribution to bacterial motility. HilD overexpression resulted in non-motile bacteria independently of flagellar assembly. It is likely that metabolic changes through collapse of the proton-motive force led to the described motility defect. Further, HilD was shown to activate expression of numerous fimbrial structures, such as Pef, Saf, or curli fimbriae. In the third chapter, alternate expression of the two antigenically distinct flagellins, FliC and FljB, was investigated regarding its importance for Salmonella physiology. FliC flagellin variants facilitated target-site selection and Spi-1 injectisome-dependent invasion during swimming on epithelial host cell surfaces. FljB-expressing bacteria were outcompeted by FliC-expressing bacteria during eukaryotic cell invasion and colonisation in the gastroenteritis mouse model. The fourth chapter focused on the posttranslational modification of the filament by the methylase FliB. FliB methylated lysine residues of the surface-exposed D2 and D3 domains of both flagellins, FliC and FljB. Moreover, flagellin methylation affected epithelial cell adhesion and invasion in a mannose-dependent manner. Consequently, strains deficient in flagellin methylation were outcompeted during dissemination in the gastroenteritis mouse model.

Many bacterial pathogens employ multicomponent protein complexes to deliver macromolecules directly into their eukaryotic host cell to promote infection. Some Gram-negative pathogens use a versatile Type IV secretion system (T4SS) that can translocate DNA or proteins into host cells. T4SSs represent major bacterial virulence determinants and have recently been the focus of intense research efforts designed to better understand and combat infectious diseases. Interestingly, although the two major classes of T4SSs function in a similar manner to secrete proteins, the translocated 'effectors' vary substantially from one organism to another. In fact, differing effector repertoires likely contribute to organism-specific host cell interactions and disease outcomes. In this review, we discuss the current state of T4SS research, with an emphasis on intracellular bacterial pathogens of humans and the diverse array of translocated effectors used to manipulate host cells.

Modulation of host cell metabolism by T4SS-encoding intracellular pathogens

The acquisition of iron is important for the growth and survival of bacterial pathogens during an infection. The majority of iron in the host is complexed in the form of the heme-containing protein, hemoglobin. To successfully cause an infection in the mammalian host, many pathogenic bacteria have developed iron acquisition systems to utilize host iron. The Gram-positive anaerobic bacterium, C. perfringens, causes human diseases including food poisoning and the potentially fatal disease, gas gangrene. In this study, a derivative of C. perfringens strain 13, JIR325, was shown to be capable of utilizing human hemoglobin and ferric chloride for its growth in vitro. Two putative heme acquisition systems, designated as the Afu and Isd systems, were investigated. The Isd system comprised of eight genes that are co-transcribed and encoded two putative surface proteins containing NEAr-iron Transporter (NEAT) domains (IsdA and IsdB), a sortase (Srt) and a membrane transport complex. The NEAT domain is a conserved region found in Gram-positive pathogens and is implicated in heme binding. Many proteins containing NEAT domains are hemoprotein receptors, which are anchored to the bacterial cell surface by membrane-anchored transpeptidases, known as sortases. Western blot analysis of IsdA,IsdB and Srt showed an increase expression of these proteins in JIR325 under iron-limited conditions. Absorbance spectroscopy and LC-MS analysis indicated that both IsdA and IsdB binds to heme. Additionally, the localization of IsdA, IsdB and Srt to the cell envelope of JIR325 suggests that IsdA and IsdB are cell wall-anchored heme-binding proteins. Growth studies of isdAisdB (isdAB) and srt mutants indicated that the isd locus was not essential for hemoglobin utilization in JIR325. The isdAB and srt mutants were attenuated for virulence in a mouse myonecrosis model, although this phenotype could not be restored with the respective complemented strains. The Afu system consisted of three genes that were co-transcribed and potentially encoded a periplasmic binding protein (AfuA), a permease (AfuB) and an ATPase (AfuC). Upstream of the afu locus is the tlyC gene, which encoded a potential hemolysin. Hemolysins lyse red blood cells to mediate the release of intracellular heme-containing proteins. Analysis of a tlyC mutant for hemolysis on blood agar suggested that tlyC does not contribute to the hemolytic activity of JIR325, under the conditions tested. Growth studies of an afuB mutant suggested that the Afu system was not essential for hemoglobin utilization. The afuB mutant was defective in growth in the presence of ferric chloride, although this phenotype could not be complemented in trans. These studies provided evidence for functional redundancies of iron and heme acquisition systems, which suggest that iron is important to the survival of JIR325.

Enhanced arginine biosynthesis and lower proteolytic profile as indicators of Saccharomyces cerevisiae stress in stationary phase during fermentation of high sugar grape must: A proteomic evidence

A-B types of toxins are among the most potent bacterial protein toxins produced by gram-positive bacteria. Prominent examples are the tripartite anthrax toxin of Bacillus anthracis and the different A-B type clostridial toxins that are the causative agents of severe human and animal diseases and could serve as biological weapons. The components of all these toxins comprise one binding/transport (B) subunit and one or two separate, non-linked enzymatically active (A) subunits. The A and B subunits are separately produced and secreted by the pathogenic gram-positive bacteria and must assemble on the surface of eukaryotic target cells to form biologically active toxin complexes. The B components are cleaved by proteases to generate the biologically active species that binds to receptors on the surface of the target cells and form there oligomers which bind the A subunits. The AB complexes are internalized by receptor-mediated endocytosis and reach early or late endosomes that become acidified. Subsequently, the B components form channels in endosomal membranes that are indispensable for the transport of the enzymatic subunits across these membranes into the cytosol of target cells via the trans-membrane channels. In addition to the channels formed by the B components, host cell factors including chaperones and further folding helper enzymes are involved in the import of the enzymatic subunits into the cytosol of eukaryotic cells. Positively charged heterocyclic molecules, such as chloroquine and related aminoquinolinium and azolopyridinium salts have been shown in recent years to bind with high affinity to the channels formed by the B components of binary toxins. Since binding to the B components is also a prerequisite for transport of the A components across the endosomal membranes the channel blockers also prevent transport of the A subunits into the host cell cytosol. The inhibition of toxin uptake into cells by such pharmacological compounds should also be of clinically interest because the toxins are the major virulence factors causing anthrax on the one hand and severe enteric disease on the other hand. Therefore, the novel toxin inhibitors should be attractive compounds for an application in combination with antibiotics to prevent or treat the diseases associated with binary toxins. Here the different processes involved in channel block in vitro and inhibition of intoxication of living target cells are reviewed in some detail.