Chlamydial development is blocked in host cells transfected with <Emphasis Type="Italic">Chlamydophila caviae incA</Emphasis>

Background  

Chlamydiae produce a set of proteins, termed Inc proteins, that are localized to the inclusion membrane and exposed to the host cell cytosol. Little information exists regarding the interaction of Inc proteins with the eukaryotic cell. To examine these interactions, Vaccinia virus vectors and mammalian plasmid-based systems were used to express inc genes in mammalian cells.     

New insights into Chlamydia intracellular survival mechanisms

Chlamydia sp. are responsible for a wide range of diseases of significant clinical and public health importance. In this review, we highlight how recent cellular and functional genomic approaches have significantly increased our knowledge of the pathogenic mechanisms used by these genetically intractable bacteria. As the extensive repertoire of chlamydial proteins that are translocated into the mammalian host is identified and characterized, a molecular understanding of how Chlamydiae co-opt host cellular functions and block innate immune pathways is beginning to emerge.     

Exotoxin secretion: getting out to find the way in

During infection, most pathogenic bacteria deliver proteins to the host cell cytoplasm to manipulate host behavior. In this issue of Cell Host & Microbe, Spanò and colleagues describe a system where a bacterium produces an exotoxin while inside the host cell. Only after this exotoxin is transported to the mammalian cell surface and secreted into the extracellular milieu can it intoxicate the infected cell or noninfected distant cells.     

Hijacking of eukaryotic functions by intracellular bacterial pathogens.

Intracellular bacterial pathogens have evolved as a group of microorganisms endowed with weapons to hijack many biological processes of eukaryotic cells. This review discusses how these pathogens perturb diverse host cell functions, such as cytoskeleton dynamics and organelle vesicular trafficking. Alteration of the cytoskeleton is discussed in the context of the bacterial entry process (invasion), which occurs either by activation of membrane-located host receptors ("zipper" mechanism) or by injection of bacterial proteins into the host cell cytosol ("trigger" mechanism). In addition, the two major types of intracellular lifestyles, cytosolic versus intravacuolar (phagosomal), which are the consequence of alterations in the phagosome-lysosome maturation route, are compared. Specific examples illustrating known mechanisms of mimicry or hijacking of the host target are provided. Finally, recent advances in phagosome proteomics and genome expression in intracellular bacteria are described. These new technologies are yielding valuable clues as to how these specialized bacterial pathogens manipulate the mammalian host cell.     

A directed screen for chlamydial proteins secreted by a type III mechanism identifies a translocated protein and numerous other new candidates

Chlamydiae are strict intracellular parasites that induce their internalization upon contact with the host cell and grow inside an intracellular compartment called an inclusion. They possess a type III secretion (TTS) apparatus, which allows for the translocation of specific proteins in the host cell cytosol. In particular, chlamydial proteins of the Inc family are secreted to the inclusion membrane by a TTS mechanism; other TTS substrates are mostly unknown. Using a secretion assay based on the recognition of TTS signals in Shigella flexneri, we searched for TTS signals in the proteins of unknown function, conserved between three different chlamydial species, Chlamydia pneumoniae, C. trachomatis and C. caviae. We identified 24 new candidate proteins which did not belong to the Inc family. Four of these proteins were also secreted as full-length proteins by a TTS mechanism in S. flexneri, indicating that their translocation does not require other chlamydial proteins. One of these proteins was detected in the cytosol of infected cells using specific antibodies, directly demonstrating that it is translocated in the host cell during bacterial proliferation. More generally, this work represents the first directed search for TTS effectors not based on genetic information or sequence similarity. It reveals the abundance of proteins secreted in the host cell by chlamydiae.     

Bacterial remodelling of the host epigenome: functional role and evolution of effectors methylating host histones

The modulation of the chromatin organization of eukaryotic cells plays an important role in regulating key cellular processes including host defence mechanisms against pathogens. Thus, to successfully survive in a host cell, a sophisticated bacterial strategy is the subversion of nuclear processes of the eukaryotic cell. Indeed, the number of bacterial proteins that target host chromatin to remodel the host epigenetic machinery is expanding. Some of the identified bacterial effectors that target the chromatin machinery are ‘eukaryotic‐like’ proteins as they mimic eukaryotic histone writers in carrying the same enzymatic activities. The best‐studied examples are the SET domain proteins that methylate histones to change the chromatin landscape. In this review, we will discuss SET domain proteins identified in the Legionella, Chlamydia and Bacillus genomes that encode enzymatic activities targeting host histones. Moreover, we discuss their possible origin as having evolved from prokaryotic ancestors or having been acquired from their eukaryotic hosts during their co‐evolution. The characterization of such bacterial effectors as modifiers of the host chromatin landscape is an exciting field of research as it elucidates new bacterial strategies to not only manipulate host functions through histone modifications but it may also identify new modifications of the mammalian host cells not known before.     

Yeast functional genomic screens lead to identification of a role for a bacterial effector in innate immunity regulation

Numerous bacterial pathogens manipulate host cell processes to promote infection and ultimately cause disease through the action of proteins that they directly inject into host cells. Identification of the targets and molecular mechanisms of action used by these bacterial effector proteins is critical to understanding pathogenesis. We have developed a systems biological approach using the yeast Saccharomyces cerevisiae that can expedite the identification of cellular processes targeted by bacterial effector proteins. We systematically screened the viable yeast haploid deletion strain collection for mutants hypersensitive to expression of the Shigella type III effector OspF. Statistical data mining of the results identified several cellular processes, including cell wall biogenesis, which when impaired by a deletion caused yeast to be hypersensitive to OspF expression. Microarray experiments revealed that OspF expression resulted in reversed regulation of genes regulated by the yeast cell wall integrity pathway. The yeast cell wall integrity pathway is a highly conserved mitogen-activated protein kinase (MAPK) signaling pathway, normally activated in response to cell wall perturbations. Together these results led us to hypothesize and subsequently demonstrate that OspF inhibited both yeast and mammalian MAPK signaling cascades. Furthermore, inhibition of MAPK signaling by OspF is associated with attenuation of the host innate immune response to Shigella infection in a mouse model. These studies demonstrate how yeast systems biology can facilitate functional characterization of pathogenic bacterial effector proteins.     

Unity in variety--the pan-genome of the Chlamydiae

Chlamydiae are evolutionarily well-separated bacteria that live exclusively within eukaryotic host cells. They include important human pathogens such as Chlamydia trachomatis as well as symbionts of protozoa. As these bacteria are experimentally challenging and genetically intractable, our knowledge about them is still limited. In this study, we obtained the genome sequences of Simkania negevensis Z, Waddlia chondrophila 2032/99, and Parachlamydia acanthamoebae UV-7. This enabled us to perform the first comprehensive comparative and phylogenomic analysis of representative members of four major families of the Chlamydiae, including the Chlamydiaceae. We identified a surprisingly large core gene set present in all genomes and a high number of diverse accessory genes in those Chlamydiae that do not primarily infect humans or animals, including a chemosensory system in P. acanthamoebae and a type IV secretion system. In S. negevensis, the type IV secretion system is encoded on a large conjugative plasmid (pSn, 132 kb). Phylogenetic analyses suggested that a plasmid similar to the S. negevensis plasmid was originally acquired by the last common ancestor of all four families and that it was subsequently reduced, integrated into the chromosome, or lost during diversification, ultimately giving rise to the extant virulence-associated plasmid of pathogenic chlamydiae. Other virulence factors, including a type III secretion system, are conserved among the Chlamydiae to variable degrees and together with differences in the composition of the cell wall reflect adaptation to different host cells including convergent evolution among the four chlamydial families. Phylogenomic analysis focusing on chlamydial proteins with homology to plant proteins provided evidence for the acquisition of 53 chlamydial genes by a plant progenitor, lending further support for the hypothesis of an early interaction between a chlamydial ancestor and the primary photosynthetic eukaryote.     

Wolbachia genomes: revealing the biology of parasitism and mutualism

Wolbachia bacteria are endosymbiotic partners of many animal species, in which they behave as either parasites (in arthropod hosts) or mutualists (in nematode hosts). What biochemistry and biology underpin these diverse lifestyles? The recent complete sequencing of genomes from Wolbachia that infect the arthropod Drosophila melanogaster and the nematode Brugia malayi, together with the partial genome sequencing of three Wolbachia strains found in drosophilids, enables this question to begin to be addressed. Parasitic arthropod Wolbachia are characterized by the presence of phages that carry ankyrin-repeat proteins; these proteins might be exported to the host cell to manipulate reproduction. In nematode Wolbachia, which lack these phages, several biochemical pathways can deliver essential metabolites to the nematode hosts. Nematode Wolbachia might also have a role in modulating the mammalian host immune system but the sequenced Wolbachia genomes lack the genes to synthesize lipopolysaccharide, raising questions about the nature of the inducing molecule. The Wolbachia surface protein might carry out this function.     

Solving a sticky problem: new genetic approaches to host cell adhesion by the Lyme disease spirochete

The Lyme disease spirochetes, comprised of at least three closely related species, Borrelia burgdorferi, Borrelia garinii and Borrelia afzelii, are fascinating and enigmatic bacterial pathogens. They are maintained by tick-mediated transmission between mammalian hosts, usually small rodents. The ability of these bacteria, which have relatively small genomes, to survive and disseminate in both an immunocompetent mammal and in an arthropod vector suggests that they have evolved elegant and indispensable strategies for interacting with their hosts. Recognition of specific mammalian and tick tissues is likely to be essential for successful completion of the enzootic life cycle but, given the historical difficulties in genetic manipulation of these organisms, characterization of factors promoting cell adhesion has until recently largely been confined to either the manipulation of host cells or the analysis of potential bacterial ligands in the form of recombinant proteins. These studies have led to the identification of several mammalian receptors for Lyme disease spirochetes, including glycosaminoglycans, decorin, fibronectin and integrins, as well as a tick receptor for the bacterium, and also candidate cognate bacterial ligands. Recent advances in our ability to genetically manipulate Lyme disease spirochetes, particularly B. burgdorferi, are now providing us with firm evidence that these ligands indeed do promote bacterial adherence to host cells, and with new insights into the roles of these multifacted Borrelia-host cell interactions during mammalian and arthropod infection.     

Identification of Chlamydia trachomatis CT621, a protein delivered through the type III secretion system to the host cell cytoplasm and nucleus

Chlamydiae are obligate intracellular bacteria, developing inside host cells within chlamydial inclusions. From these inclusions, the chlamydiae secrete proteins into the host cell cytoplasm. A pathway through which secreted proteins can be delivered is the type III secretion system (T3SS). The T3SS is common to several gram-negative bacteria and the secreted proteins serve a variety of functions often related to the modulation of host signalling. To identify new potentially secreted proteins, the cytoplasm was extracted from Chlamydia trachomatis L2-infected HeLa cells, and two-dimensional polyacrylamide gel electrophoresis profiles of [³⁵S]-labelled chlamydial proteins from this extract were compared with profiles of chlamydial proteins from the lysate of infected cells. In this way, CT621 was identified. CT621 is a member of a family of proteins containing a domain of unknown function DUF582 that is only found within the genus Chlamydia . Immunofluorescence microscopy and immunoblotting demonstrated that CT621 is secreted late in the chlamydial developmental cycle and that it is the first chlamydial protein found to be localized within both the host cell cytoplasm and the nucleus. To determine whether CT621 is secreted through the T3SS, an inhibitor of this apparatus was added to the infection medium, resulting in retention of the protein inside the chlamydiae. Hence, the so far uncharacterized CT621 is a new type III secretion effector protein.     

The role of cell signaling in poxvirus tropism: the case of the M-T5 host range protein of myxoma virus

Poxviruses demonstrate strict species specificity in vivo that range from narrow to broad, however the fundamental factors that mediate the basis of poxvirus tropism remain poorly understood. It is generally believed that most, if not all, poxviruses can efficiently bind and enter a wide range of mammalian cells and all of the known host anti-viral pathways that block viral replication in nonpremissive cells operate downstream of virus entry. A productive poxvirus infection is heavily dependent upon the production of a vast array of host modulatory products that specifically target and manipulate both extracellular immune response pathways of the host, as well as intracellular signal transduction pathways of the individually infected cells. The unique pathogenesis and host tropism of specific poxviruses can be attributed to the broad diversity of host modulatory proteins they express. Myxoma virus (MV) is a rabbit-specific poxviruses that encodes multiple host range factors, including an ankyrin-repeat protein M-T5, which functions to regulate tropism of MV for rabbit lymphocytes and some human cancer cells. At the molecular level, M-T5 binds and alters at least two distinct cellular proteins: Akt and cullin-1. The direct interaction between M-T5 and Akt was shown to be a key restriction determinant for MV tropism in a spectrum of human cancer cells making MV an excellent oncolytic candidate. Thus, the intricate relationship between viral encoded proteins and components of the host cell signaling networks can have profound impact on poxvirus tropism. The lessons we continue to learn from poxvirus host range factors like M-T5 will provide further insights into the factors that regulate poxvirus tropism and the mechanisms by which poxviruses micromanipulate the signaling pathways of the infected cell.     

Toxoplasma Co-opts Host Cells It Does Not Invade

Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.     

Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance

Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.     

Intracellular survival by Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens whose entry into mucosal epithelial cells is required for intracellular survival and subsequent growth. After a seemingly stealthy entry, chlamydiae quickly modify their vacuole (i) for exit from the endosomal pathway to the exocytic pathway and (ii) to permit fusion with intercepted endoplasmic reticulum- and Golgi-derived vesicles carrying glycerophospholipids and sphingolipids for chlamydiae-containing vacuole membrane expansion. Chlamydiae possess novel hollow proteinaceous structures, termed projections, which they use to pierce the inclusion membrane, possibly to acquire from the epithelial cytoplasm nutrients they cannot synthesize; whether or not these truncated flagellar-like structures serve a dual exchange function for secretion of molecules to programme host cell signalling is unknown. Despite the accumulation of some 500–1000 progeny in the enormously enlarged inclusion, host cell function is surprisingly little disrupted, and progeny escape can be unobtrusive. This elegant adaptive pathogen strategy, which leads to silent, chronic human infection, is fascinating from a cellular microbiology perspective.     

Exploiting pathogenic Escherichia coli to model transmembrane receptor signalling

Many microbial pathogens manipulate the actin cytoskeleton of eukaryotic target cells to promote their internalization, intracellular motility and dissemination. Enteropathogenic and enterohaemorrhagic Escherichia coli, which both cause severe diarrhoeal disease, can adhere to mammalian intestinal cells and induce reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria. As pedestal assembly is triggered by E. coli virulence factors that mimic several host cell-signalling components, such as transmembrane receptors, their cognate ligands and cytoplasmic adaptor proteins, it can serve as a powerful model system to study eukaryotic transmembrane signalling. Here, we consider the impact of recent data on our understanding of both E. coli pathogenesis and cell biology, and the rich prospects for exploiting these bacterial factors as versatile tools to probe cellular signalling pathways.     

Modulation of cytokines and transcription factors (T-Bet and GATA3) in CD4 enriched cervical cells of Chlamydia trachomatis infected fertile and infertile women upon stimulation with chlamydial inclusion membrane proteins B and C

Background  

Chlamydial Inclusion membrane proteins (Incs), are involved in biochemical interactions with host cells and infecting Chlamydiae. We have previously reported the role of two Chlamydia trachomatis (CT) Incs, namely IncB and IncC in generating host immunity in CT infected women. Emerging data shows involvement of Inc stimulated CD4 positive T cells in aiding host immunity in infected fertile and infertile women through the secretion of interferon gamma. However the lack of data on the intra-cytokine interplay to these Incs in infected cell milieu prompted us to investigate further.     

Immunity to vacuolar pathogens: What can we learn from Legionella?

Intracellular pathogens can manipulate host cellular pathways to create specialized organelles. These pathogen-modified vacuoles permit the survival and replication of bacterial and protozoan microorganisms inside of the host cell. By establishing an atypical organelle, intracellular pathogens present unique challenges to the host immune system. To understand pathogenesis, it is important to not only investigate how these organisms create unique subcellular compartments, but to also determine how mammalian immune systems have evolved to detect and respond to pathogens sequestered in specialized vacuoles. Recent studies have identified genes in the respiratory pathogen Legionella pneumophila that are essential for establishing a unique endoplasmic reticulum-derived organelle inside of mammalian macrophages, making this pathogen an attractive model system for investigations on host immune responses that are specific for bacteria that establish vacuoles disconnected from the endocytic pathway. This review will focus on the host immune response to Legionella and highlight areas of Legionella research that should help elucidate host strategies to combat infections by intracellular pathogens.