Chlamydial MreB Directs Cell Division and Peptidoglycan Synthesis in Escherichia coli in the Absence of FtsZ Activity.

Cell division is the ultimate process for the propagation of bacteria, and FtsZ is an essential protein used by nearly all bacteria for this function. Chlamydiae belong to a small group of bacteria that lack the universal cell division protein FtsZ but still divide by binary fission. Chlamydial MreB is a member of the shape-determining MreB/Mbl family of proteins responsible for rod shape morphology in Escherichia coliChlamydia also encodes a homolog of RodZ, an MreB assembly cytoskeletal protein that links MreB to cell wall synthesis proteins. We hypothesized that MreB directs cell division in Chlamydia and that chlamydial MreB could replace FtsZ function for cell division in E. coli Overexpression of chlamydial mreB-rodZ in E. coli induced prominent morphological changes with production of large swollen or oval bacteria, eventually resulting in bacterial lysis. Low-level expression of chlamydial mreB-rodZ restored viability of a lethal ΔmreB mutation in E. coli, although the bacteria lost their typical rod shape and grew as rounded cells. When FtsZ activity was inhibited by overexpression of SulA in the ΔmreB mutant of E. coli complemented with chlamydial mreB-rodZ, spherical E. coli grew and divided. Localization studies using a fluorescent fusion chlamydial MreB protein indicated that chlamydial RodZ directs chlamydial MreB to the E. coli division septum. These results demonstrate that chlamydial MreB, in partnership with chlamydial RodZ, acts as a cell division protein. Our findings suggest that an mreB-rodZ-based mechanism allows Chlamydia to divide without the universal division protein FtsZ.IMPORTANCE The study of Chlamydia growth and cell division is complicated by its obligate intracellular nature and biphasic lifestyle. Chlamydia also lacks the universal division protein FtsZ. We employed the cell division system of Escherichia coli as a surrogate to identify chlamydial cell division proteins. We demonstrate that chlamydial MreB, together with chlamydial RodZ, forms a cell division and growth complex that can replace FtsZ activity and support cell division in E. coli Chlamydial RodZ plays a major role in directing chlamydial MreB localization to the cell division site. It is likely that the evolution of chlamydial MreB and RodZ to form a functional cell division complex allowed Chlamydia to dispense with its FtsZ-based cell division machinery during genome reduction. Thus, MreB-RodZ represents a possible mechanism for cell division in other bacteria lacking FtsZ.

Deconstructing the Chlamydial Cell Wall.

The evolutionary separated Gram-negative Chlamydiales show a biphasic life cycle and replicate exclusively within eukaryotic host cells. Members of the genus Chlamydia are responsible for many acute and chronic diseases in humans, and Chlamydia-related bacteria are emerging pathogens. We revisit past efforts to detect cell wall material in Chlamydia and Chlamydia-related bacteria in the context of recent breakthroughs in elucidating the underlying cellular and molecular mechanisms of the chlamydial cell wall biosynthesis. In this review, we also discuss the role of cell wall biosynthesis in chlamydial FtsZ-independent cell division and immune modulation. In the past, penicillin susceptibility of an invisible wall was referred to as the "chlamydial anomaly." In light of new mechanistic insights, chlamydiae may now emerge as model systems to understand how a minimal and modified cell wall biosynthetic machine supports bacterial cell division and how cell wall-targeting beta-lactam antibiotics can also act bacteriostatically rather than bactericidal. On the heels of these discussions, we also delve into the effects of other cell wall antibiotics in individual chlamydial lineages.

Subversion of Cell-Autonomous Host Defense by Chlamydia Infection.

Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

Insight in the biology of Chlamydia-related bacteria.

The Chlamydiales order is composed of obligate intracellular bacteria and includes the Chlamydiaceae family and several family-level lineages called Chlamydia-related bacteria. In this review we will highlight the conserved and distinct biological features between these two groups. We will show how a better characterization of Chlamydia-related bacteria may increase our understanding on the Chlamydiales order evolution, and may help identifying new therapeutic targets to treat chlamydial infections.

Mito-xenophagic killing of bacteria is coordinated by a metabolic switch in dendritic cells.

Chlamydiae are bacterial pathogens that grow in vacuolar inclusions. Dendritic cells (DCs) disintegrate these compartments, thereby eliminating the microbes, through auto/xenophagy, which also promotes chlamydial antigen presentation via MHC I. Here, we show that TNF-α controls this pathway by driving cytosolic phospholipase (cPLA)2-mediated arachidonic acid (AA) production. AA then impairs mitochondrial function, which disturbs the development and integrity of these energy-dependent parasitic inclusions, while a simultaneous metabolic switch towards aerobic glycolysis promotes DC survival. Tubulin deacetylase/autophagy regulator HDAC6 associates with disintegrated inclusions, thereby further disrupting their subcellular localisation and stability. Bacterial remnants are decorated with defective mitochondria, mito-aggresomal structures, and components of the ubiquitin/autophagy machinery before they are degraded via mito-xenophagy. The mechanism depends on cytoprotective HSP25/27, the E3 ubiquitin ligase Parkin and HDAC6 and promotes chlamydial antigen generation for presentation on MHC I. We propose that this novel mito-xenophagic pathway linking innate and adaptive immunity is critical for effective DC-mediated anti-bacterial resistance.

Unexpected genomic features in widespread intracellular bacteria: evidence for motility of marine chlamydiae.

Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments.

FtsZ-independent septal recruitment and function of cell wall remodelling enzymes in chlamydial pathogens.

The nature and assembly of the chlamydial division septum is poorly defined due to the paucity of a detectable peptidoglycan (PG)-based cell wall, the inhibition of constriction by penicillin and the presence of coding sequences for cell wall precursor and remodelling enzymes in the reduced chlamydial (pan-)genome. Here we show that the chlamydial amidase (AmiA) is active and remodels PG in Escherichia coli. Moreover, forward genetics using an E. coli amidase mutant as entry point reveals that the chlamydial LysM-domain protein NlpD is active in an E. coli reporter strain for PG endopeptidase activity (ΔnlpI). Immunolocalization unveils NlpD as the first septal (cell-wall-binding) protein in Chlamydiae and we show that its septal sequestration depends on prior cell wall synthesis. Since AmiA assembles into peripheral clusters, trimming of a PG-like polymer or precursors occurs throughout the chlamydial envelope, while NlpD targets PG-like peptide crosslinks at the chlamydial septum during constriction.

Expression patterns of five polymorphic membrane proteins during the Chlamydia abortus developmental cycle.

It has been suggested that polymorphic membrane proteins (Pmps) belonging to the Type V autotransporter protein family play an important role in the pathogenesis of Chlamydia abortus (C. abortus; formerly Chlamydophila abortus) infection. In a previous study we demonstrated the expression of all the pmps at the transcriptional level. The purpose of this study was to measure the number of Pmp positive inclusions throughout the C. abortus developmental cycle to investigate heterogeneity in expression patterns. McCoy cells were infected with C. abortus and analysed for Pmp expression over a 72 h period by fluorescent immunocytochemistry. Pmp18D could be detected at all analysed time points, and could only be accurately quantified from 36 hpi while Pmp10G positive inclusions could be visualised from 36hpi. Expression of Pmps 13G, 16G and 17G could only be visualised later in the cycle and within less than half of visualised inclusions. These results indicate that while expression of specific Pmps is constitutive (Pmp18D), the pattern of expression of other Pmps is more variable. This suggests that different members of the Pmp family may play different roles within the developmental cycle of the organism, with some (Pmps10G and 18D) having roles throughout the cycle, while the heterogeneity of expression of others may aid in antigenic variation.

Planctomycetes and eukaryotes: a case of analogy not homology.

Planctomycetes, Verrucomicrobia and Chlamydia are prokaryotic phyla, sometimes grouped together as the PVC superphylum of eubacteria. Some PVC species possess interesting attributes, in particular, internal membranes that superficially resemble eukaryotic endomembranes. Some biologists now claim that PVC bacteria are nucleus-bearing prokaryotes and are considered evolutionary intermediates in the transition from prokaryote to eukaryote. PVC prokaryotes do not possess a nucleus and are not intermediates in the prokaryote-to-eukaryote transition. Here we summarise the evidence that shows why all of the PVC traits that are currently cited as evidence for aspiring eukaryoticity are either analogous (the result of convergent evolution), not homologous, to eukaryotic traits; or else they are the result of horizontal gene transfers.

Raman microspectroscopy reveals long-term extracellular activity of Chlamydiae.

The phylum Chlamydiae consists exclusively of obligate intracellular bacteria. Some of them are formidable pathogens of humans, while others occur as symbionts of amoebae. These genetically intractable bacteria possess a developmental cycle consisting of replicative reticulate bodies and infectious elementary bodies, which are believed to be physiologically inactive. Confocal Raman microspectroscopy was applied to differentiate between reticulate bodies and elementary bodies of Protochlamydia amoebophila and to demonstrate in situ the labelling of this amoeba symbiont after addition of isotope-labelled phenylalanine. Unexpectedly, uptake of this amino acid was also observed for both developmental stages for up to 3 weeks, if incubated extracellularly with labelled phenylalanine, and P. amoebophila remained infective during this period. Furthermore, P. amoebophila energizes its membrane and performs protein synthesis outside of its host. Importantly, amino acid uptake and protein synthesis after extended extracellular incubation could also be demonstrated for the human pathogen Chlamydia trachomatis, which synthesizes stress-related proteins under these conditions as shown by 2-D gel electrophoresis and MALDI-TOF/TOF mass spectrometry. These findings change our perception of chlamydial biology and reveal that host-free analyses possess a previously not recognized potential for direct experimental access to these elusive microorganisms.

[Chlamydia: past, present and future].

In this overview is observed the history and presence of Chlamydia infections, which accompanied the human in its evolution. As a sexually transmitted disease it affects the fertile age. The unique life-cycle of this intra-cellular microorganism is reason for the difficulties of its identification in the past. The technological development in immunology and microbiology field, in present days made possible its isolation, antigen analysis and antibodies identification in the infected person and as well as serology typification. This allows to diagnose and treat in due time and secondary prophylaxis as well. There is no perfect laboratory test. Humans do not possess natural immunity against Chlamydia and at the same time the infection does not lead to durable immunity.

Proteins in the chlamydial inclusion membrane.

The chlamydiae are obligate intracellular pathogens that occupy a nonacidified vacuole (the inclusion) during their entire developmental cycle. Several proteins have recently been identified that are localized to the inclusion membrane. The following is a discussion of how inclusion membrane proteins might participate in the chlamydial developmental process.

[Comparative analysis of methods of diagnosis of chlamydia infection]. / Sravnitel'nyi analiz metodov diagnostiki khlamidioza.

In case of a correct sampling, the diagnostic value of optic and electron microscopy for detecting Chlamydia infection is not inferior to that of direct microimmunofluorescence (DMIF) and higher than that of enzyme immunoassay (EIA). Optic microscopy showed that basal vaginal epithelium and buccal mucosa can be infected with Chlamydia. Provazek bodies were detected in the buccal mucosa of the overwhelming majority of patients with genital chlamydiasis. These results were confirmed by DMIF and EIA. Since none of the diagnostic methods is 100% reliable, we recommend using two methods: inexpensive optic microscopy and polymerase chain reaction or DMIF.

Epitheliocystis agents in sea bream Sparus aurata: morphological evidence for two distinct chlamydia-like developmental cycles.

The morphology of membrane-bound intracellular inclusions, or 'cysts', of epitheliocystis from sea bream Sparus aurata is described. Inclusions under the light microscope appear either granular or amorphous. Granular inclusions do not elicit a proliferative host reaction and contain the 3 distinctive developmental stages of chlamydial organisms: the highly pleomorphic reproductive form or reticulate body, the condensing form or intermediate body and the infective non-dividing rather uniform elementary body. Amorphous inclusions may elicit a proliferative host reaction and contain prokaryotic organisms which differ morphologically from those reported within granular cysts. More or less elongated electron-lucent organisms divide by fission to give rise to electron-dense non-dividing small cells with a dense nucleoid. Vacuolated and non-vacuolated small cells are reported. The morphology and developmental cycle of sea bream epitheliocystis agents would support their chlamydial nature; however, the immunohistochemical study conducted on gill samples which carried both inclusions failed to demonstrate the expression of lipopolysaccharide (LPS) chlamydial antigen. The different stages of the 2 distinct developmental cycles described in the present study are compared with electron microscope observations of epitheliocystis organisms reported from different host species. The hypothesis that epitheliocystis infection in the sea bream might be caused by a unique highly pleomorphic chlamydia-like agent, the life history of which includes 2 entirely different developmental cycles, is discussed.

Pneumonia caused by Mycoplasma pneumoniae and the TWAR agent.

Mycoplasma pneumoniae and the TWAR agent account for a good proportion of pneumonias acquired in the community among older children and young adults. Recovery from these infections is common, although serious complications may occur. Each is associated with fever, nonproductive cough, and headache. The full clinical manifestations of TWAR agent infection are only now beginning to be defined. Diagnosis of M pneumoniae can be by serology and/or culture capabilities. The TWAR agent cannot be routinely isolated and serologic tests are available only in research laboratories. Response to tetracycline antibiotics has been established for each agent and hospitalization is rarely needed. Erythromycin and other macrolide antibiotics have good activity against M pneumoniae and can be employed as an alternative to tetracycline antibiotics for this pathogen.

Rickettsiaceae and Chlamydiaceae: comparative electron microscopic studies.

The structure and cytopathology of obligate intracellular bacteria belonging to families Rickettsiaceae and Chlamydiaceae and their interaction with eukaryotic host cells were compared in electron microscopic studies. "Rickettsia-like" and "chlamydia-like" types of organization of bacterial cells and their interaction with host cells are presented. The rickettsia-like type is characterized by short rod-shaped cells multiplying freely ( extravacuolarly ) in the cytoplasm or nucleoplasm of the host cell; the chlamydia-like type has spherical cells multiplying inside the cytoplasmic vacuole limited by the host membrane. The rickettsia-like type includes the genus Rickettsia and rod-shaped symbionts from genera Wolbachia and Symbiotes ; the chlamydia-like type falls into genera Chlamydia, Ehrlichia, Cowdria and Neorickettsia . The transitional types represented by Wolbachia persica (type 1), Coxiella and Rickettsiella (type 2) are also described. The possible evolutional relationships of the genera comprising both families are considered and their classification is proposed.

Demonstration of chlamydial inclusions in exfoliated cells.

Microscopic demonstration of chlamydial inclusions within cells offered the first laboratory procedure supporting the clinical diagnosis of chlamydial infection. The cytological test for the detection of chlamydial inclusions in genital tract infection, through not as sensitive as isolation in cell cultures, is still of diagnostic value. The present study discusses the collection of clinical specimens for microscopic examination, the preparation, fixation and staining of slides by Giemsa stain, iodine, and immunofluorescence.

[Procedure of standardization of Chlamydiae suspensions (author's transl)]. / Une méthode de standardisation des suspensions Chlamydiennes.

The enumeration of a chlamydiae suspension can be achieved by the addition of a standardized shigella suspension; the two cell populations, after coloration with acridine, are counted in microdrops. This procedure permits to plot a turbidity curve.

[Unusual small forms in the growth cycle of halprowia (chlamydia) and their possible relation to manifestations of L-transformation]. / Neobychnye melkie formy v tsikle rezvitiia gal'provii (khlamidii) i ikh vozmozhnaia sviaz' s iavleniiami L-transformatsii

The authors carried out a comparative electron-microscopic study of the ultrastructure and the developmental cycle of 15 Halprowiae (chlamydiae) strains isolated in different pathology from man and animals. Halprowiae were cultivated in the yolk sac epithelium of the developing chick embryos and in the L-cell monolayer cultures without any special action of the antibiotics and medicines. Formation of minute round structures ("minute forms"), 50--120 nm in diameter, separating from the protoplast into the periplasmic space and surrounded with cytoplasmic membrane by the vegetative forms of a number of strains (MP, AP-23, 25-SM, LB-1, MyB), is described. Along with minute forms, Halprowiae formed polymorphous giant bodies with long protrusions into the inclusion cavity; there was also a separation of vesicles of the cell wall membrane. This apparently indicated a structural derangement of the cell wall in these vegetative forms of Halprowiae. On the basis of similarity of the mentioned structures detected in Halprowiae reproduction to the known morphological characteristics of the L-form bacteria a possibility of L-transformation in Halprowiae during the normal developmental cycle as a peculiar compensatory adaptation to the action of host cell is supposed.