Bacterial Type IV Secretion Systems: Versatile Virulence Machines

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 complex interactions of bacterial pathogens and host defenses

Staphylococcus aureus is a common bacterial etiology of serious infectious diseases. S. aureus can invade various types of non-professional phagocytes to produce host cell death. We show here that shortly after invasion of HeLa cells S. aureus transit to autophagosomes was characterized by double membranes and co-localization with LC3. S. aureus were not able to replicate and produce cell death in autophagy-deficient atg5-/- mouse embryonic fibroblasts. S. aureus-containing autophagosomes do not acidify nor do they acquire lysosome-associated membrane protein-2, indicating that S. aureus inhibits autophagosome maturation and fusion with lysosomes. Eventually, S. aureus escape from autophagosomes into the cytoplasm, which results in caspase-independent host cell death. S. aureus strains deficient for agr, a global regulator of S. aureus virulence, were not targeted by autophagy and did not produce host-cell death. Autophagy induction by rapamycin restored both replication and cytotoxicity of agr-deficient S. aureus strains, indicating that an agr-regulated factor(s) is required for autophagy-mediated cytotoxicity. The results of this study suggest that rapid induction of autophagy is essential for S. aureus replication, escape into the cytoplasm, and host cell killing.

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.

The Type Three Secretion (T3S) system is a device used by many Gram-negative pathogens that allows bacteria to deliver effector proteins straight into the eukaryotic cell cytosol. These effectors interfere with various signaling pathways to subvert the host cell functions. The secretion machinery of the T3S system consist of a basal body spanning the bacterial inner and outer membrane followed by a stiff hollow needle outside the bacterium. The fully assembled secretion apparatus constitute a continuous hollow conduit that connects the bacteria to the eukaryotic target cell. After cell contact, virulence proteins -called effectors- are injected directly into the cytosol of the host cell via the T3S apparatus. Several effectors of the T3S system require the assistance of specific cytosolic chaperones to be efficiently exported. There are three classes of T3S chaperones. Effector proteins are assisted by Class I chaperones. Although Class I chaperones are well characterized, their main function is still a matter of controversy. In this thesis, we demonstrate that orf155 encodes a specific chaperone for the effector YopO that we called SycO. We showed that SycO enhances YopO secretion in vitro and is required for translocation of YopO into infected cells. By pulldown assay we demonstrated that residues 20 to 77 of YopO are required and sufficient for SycO binding. Using crosslinking experiments and size exclusion chromatography analysis, we determined the stoichiometry of purified SycO and YopO-SycO complexes. SycO alone forms dimers in solution and the YopO-SycO complex has a 1:2 stoichiometry. These results suggested that SycO is a typical chaperone of the Class I. YopO is a serine/theronine kinase that interacts with Rho and Rac and disrupts the cytoskeleton of the target cells. YopO has been shown to localize at the cell plasma-membrane. By transfection of YopO-EGFP hybrid proteins into HEK293T cells, we demonstrated that the chaperone-binding domain (CBD) coincides with the membrane localization domain of YopO. Nevertheless, the CBD was not needed for the kinase activity of YopO. By ultracentrifugation, we also showed that the CBD causes YopO aggregation in the bacteria, when SycO does not cover it. Further, we show that the CBD of YopE and YopT also caused aggregation in the bacteria in the absence of SycE and SycT respectively. YopE, YopT and T3S effectors in other systems also act at the membrane of the eukaryotic host cell. We propose a new hypothesis concerning the role of T3S chaperones. The sub-cellular localization domain of effectors is aggregation-prone and creates the need for a chaperone inside bacteria. We propose that masking such aggregation-prone localization domains may be a general function for type III effector chaperones.

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.

### 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

Bacterial effector interplay: a new way to view effector function

Enteropathogenic Escherichia coli (EPEC) is a gram-negative bacterial pathogen that adheres to human intestinal epithelial cells, resulting in watery, persistent diarrhea. It subverts the host cell cytoskeleton, causing a rearrangement of cytoskeletal components into a characteristic pedestal structure underneath adherent bacteria. In contrast to other intracellular pathogens that affect the actin cytoskeleton from inside the host cytoplasm, EPEC remains extracellular and transmits signals through the host cell plasma membrane via direct injection of virulence factors by a "molecular syringe," the bacterial type III secretion system. One injected factor is Tir, which functions as the plasma membrane receptor for EPEC adherence. Tir directly links extracellular EPEC through the epithelial membrane and firmly anchors it to the host cell actin cytoskeleton, thereby initiating pedestal formation. In addition to stimulating actin nucleation and polymerization in the host cell, EPEC activates several other signaling pathways that lead to tight junction disruption, inhibition of phagocytosis, altered ion secretion, and immune responses. This review summarizes recent developments in our understanding of EPEC pathogenesis and discusses similarities and differences between EPEC pedestals, focal contacts, and Listeria monocytogenes actin tails.

Identification of a Molecular Target for the Yersinia Protein Kinase A

This chapter discuss three critical aspects of the interaction of Campylobacter jejuni with host cells: (i) its ability to mediate its own uptake into nonphagocytic cells, (ii) its ability to modulate vesicular trafficking pathways to avoid delivery into lysosomes, and (iii) its ability to reprogram host cell gene expression to stimulate the production of proinflammatory cytokines. The strong phenotype associated with capsular mutants in different animal models is likely to be due to reasons other than their rather minor effect in bacterial internalization. Therefore, it is likely that the capsular polysaccharide may contribute to internalization indirectly, perhaps by promoting bacterial attachment to host cells. Bacterial internalization does not require dynamin, an essential component of the endocytic machinery associated with cavaealoe. Like other intracellular pathogens, C. jejuni must have evolved specific adaptations to survive within host cells. Intestinal epithelial cells are equipped to mount innate immune responses upon detection of microbial pathogens. The last few years have seen advances in the understanding of the cell biology of infection, although more studies will be required to gain an understanding of these issues on par with that of other pathogenic bacteria. Although many C. jejuni mutants apparently defective in some of these process have been identified (e.g., bacterial entry), the direct involvement of these determinants in C. jejuni– host cell interactions has not been demonstrated. The availability of powerful genetic tools, coupled with a better understanding of the cell biology of infection, can help to identify those bacterial determinants.

Francisella tularensis is a zoonotic pathogen and the causative agent of tularemia. F. tularensis replicates to high levels within the cytosol of macrophages and other host cells while subverting the host response to infection. Critical to the success of F. tularensis is its ability to delay macrophage apoptosis to maintain its intracellular replicative niche. However, the host-signaling pathway(s) modulated by F. tularensis to delay apoptosis are poorly characterized. The outer membrane channel protein TolC is required for F. tularensis virulence and its ability to suppress apoptosis and cytokine expression during infection of macrophages. We took advantage of the F. tularensis ∆tolC mutant phenotype to identify host pathways that are important for activating macrophage apoptosis and that are disrupted by the bacteria. Comparison of macrophages infected with wild-type or ∆tolC F. tularensis revealed that the bacteria interfere with TLR2-MYD88-p38 signaling at early times post infection to delay apoptosis, dampen innate host responses, and preserve the intracellular replicative niche. Experiments using the mouse pneumonic tularemia model confirmed the in vivo relevance of these findings, revealing contributions of TLR2 and MYD88 signaling to the protective host response to F. tularensis, which is modulated by the bacteria to promote virulence. IMPORTANCE Francisella tularensis is a Gram-negative intracellular bacterial pathogen and the causative agent of the zoonotic disease tularemia. F. tularensis, like other intracellular pathogens, modulates host-programmed cell death pathways to ensure its replication and survival. We previously identified the outer membrane channel protein TolC as required for the ability of F. tularensis to delay host cell death. However, the mechanism by which F. tularensis delays cell death pathways during intracellular replication is unclear despite being critical to pathogenesis. In the present study, we address this gap in knowledge by taking advantage of ∆tolC mutants of F. tularensis to uncover signaling pathways governing host apoptotic responses to F. tularensis and which are modulated by the bacteria during infection to promote virulence. These findings reveal mechanisms by which intracellular pathogens subvert host responses and enhance our understanding of the pathogenesis of tularemia.

Coxiella burnetii is an obligate intracellular gram-negative bacterial pathogen, an ethiological agent of Q-fever, a zoonotic disease, elapsing as an acute (mostly atypical pneumonia) or a chronic (mostly endocarditis) form. The host range is represented by wide range of mammal, avian and arthropod species, but the main source of human infection are farm animals. The main route of infection is aerosolic. In case of contact with organism pathogen binds with phagocytal monocytic-macrophagal cell line. C. burnetii promotes maturation of specific phagolysosome-like compartment in host cell, called coxiella-containing vacuole, within this vacuole pathogen becames metabolically activated and actively replicates. Coxiella persists as metabolically inactive spore-like form in environment. Internalisation of C. burnetii occurs using actin-mediated phagocytosis and zipper mechanism. After internalization of bacteria maturation of phagolysosome-like compartment and large coxiella-containing vacuole formation occure, and vacuole can occupy nearly the whole cytoplasm of the host cell. Survivance of infected cells is important for chronic infection with C. burnetii. C. burnetii elongate the viability of host cell by two ways: it actively inhibits apoptotic signal cascades and induce pro-survival factors. Exceptthat C. burnetii involves autophagic pathway during coxiella-containing vacuole formation, and induction of autophagy promotes pathogen replication. During infection C. burnetii translocates effector substrates from bacterial cytosole to euca ryotic host cell cytosole using type IV secretion system, where effectors modulate host cell proteins. Overall approximately 130 secreted effectors of type IV transport system, but function of most of them remains unknown to date. Specific sec reted proteins for variety of strains and isolates were identified, confirmed that certain pathotypes of C. burnetii can exist. Identification and characterization of novel virulence factors it is now possible through axenic media for C. burnetii cultivation and development of site-specific mutagenesis and other genetic technics, which is important for research of C. burnetii molecular pathogenesis.

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.

Bacterial pathogens subvert host cells by using their gene products to manipulate cellular pathways for survival and replication; in turn, host cells respond to invading pathogens through cascading changes in gene expression. Deciphering this complex temporal and spatial interplay is crucial for improved diagnostics and therapeutics. However, the technical limitations of existing methods of expression profiling, such as microarray analysis, significantly reduce their utility for obtaining both bacterial and host expression profiles from infected cells. We developed the heterogeneous RNA-Seq (hRNA-Seq) method to simultaneously capture prokaryotic and eukaryotic expression profiles of bacteria-infected cells. As proof of principle, hRNA-Seq was applied to <em>Chlamydia</em>-infected cells, successfully obtaining the transcriptomes of both <em>Chlamydia</em> and their host cells at 1 and 24 hours post-infection. Substantial transcription was found in the immediate-early period of infection for both <em>Chlamydia</em> and the host cell. Numerous novel host responses were found; in particular we discovered putative positive feedback mechanisms for <em>Chlamydia</em>-induced fibrotic scarring.