Obligate Intracellular Lifestyle Research Articles

Chlamydiae in corals: shared functional potential despite broad taxonomic diversity.

Cnidarians, such as corals and sea anemones, associate with a wide range of bacteria that have essential functions, including nutrient cycling and the production of antimicrobial compounds. Within cnidarians, bacteria can colonize all microhabitats including the tissues. Among them are obligate intracellular bacteria of the phylum Chlamydiota (chlamydiae) whose impact on cnidarian hosts and holobionts, especially corals, remain unknown. Here, we conducted a meta-analysis of previously published 16S rRNA gene metabarcoding data from cnidarians (e.g. coral, jellyfish, and anemones), eight metagenome-assembled genomes (MAGs) of coral-associated chlamydiae, and one MAG of jellyfish-associated chlamydiae to decipher their diversity and functional potential. While the metabarcoding dataset showed an enormous diversity of cnidarian-associated chlamydiae, six out of nine MAGs were affiliated with the Simkaniaceae family. The other three MAGs were assigned to the Parasimkaniaceae, Rhabdochlamydiaceae, and Anoxychlamydiaceae, respectively. All MAGs lacked the genes necessary for an independent existence, lacking any nucleotide or vitamin and most amino acid biosynthesis pathways. Hallmark chlamydial genes, such as a type III secretion system, nucleotide transporters, and genes for host interaction, were encoded in all MAGs. Together these observations suggest an obligate intracellular lifestyle of coral-associated chlamydiae. No unique genes were found in coral-associated chlamydiae, suggesting a lack of host specificity. Additional studies are needed to understand how chlamydiae interact with their coral host, and other microbes in coral holobionts. This first study of the diversity and functional potential of coral-associated chlamydiae improves our understanding of both the coral microbiome and the chlamydial lifestyle and host range.

Orientia tsutsugamushi: comprehensive analysis of the mobilome of a highly fragmented and repetitive genome reveals the capacity for ongoing lateral gene transfer in an obligate intracellular bacterium.

Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

Knock-sideways by inducible ER retrieval enables a unique approach for studying Plasmodium-secreted proteins

Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting Plasmodium-secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple Plasmodium species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.

Telomere-to-Telomere genome assemblies of human-infecting Encephalitozoon species

BackgroundMicrosporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete.MethodsIn this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. —E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602— using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation.ResultsThe Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes.ConclusionAltogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

Transformation and Mutagenesis of Chlamydia trachomatis and C. muridarum Utilizing pKW Vector

A gene deletion by allelic exchange via homologous recombination from a bacterial genome represents a valuable genetic tool for studying a role(s) of determinants involved in various aspects of pathogenesis. Due to chlamydial obligate intracellular lifestyle and comparatively low transformation rate, the mutagenesis of Chlamydia utilizes types of suicide vectors that have to be maintained and propagated by the bacteria throughout several rounds of their intracellular developmental cycle. These deletion constructs must be lost by chlamydiae once null mutant formation is achieved. pKW is a small, 5.45 bp, pUC19-derived vector, which has been recently successfully employed for the generation of deletion mutants in C. trachomatis, serovariant D, and C. muridarum. This vector contains both, E. coli as well as chlamydial species-specific plasmid origins of replication, allowing for its propagation by both bacterial genera under a selective pressure. However, once the selective antibiotic is removed from culture, chlamydiae rapidly lose pKW, and the subsequent reintroduction of the selective antibiotic back to chlamydiae-infected cells results efficiently in the selection of generated deletion mutants. Protocols provided here describe in detail the preparation of pKW deletion constructs for C. trachomatis and C. muridarum applicable for chlamydial transformation and the production of null mutant in non-essential genes. Protocols provided here, are describing in detail methods for assembly of the pKW shuttle vector and generation of deletion mutants in C. trachomatis and C. muridarum. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Assembly of pKW shuttle vector Basic Protocol 2: Generation of a deletion mutant in C. trachomatis, serovars D and L2 and, Chlamydia muridarum Support Protocol: Transformation of C. trachomatis, serovars B.

Quantitative analysis of morphogenesis and growth dynamics in an obligate intracellular bacterium.

Obligate intracellular bacteria of the order Rickettsiales include important human pathogens. However, our understanding of the biology of Rickettsia species is limited by challenges imposed by their obligate intracellular lifestyle. To overcome this roadblock, we developed methods to assess cell wall composition, growth, and morphology of Rickettsia parkeri, a human pathogen in the spotted fever group of the Rickettsia genus. Analysis of the cell wall of R. parkeri revealed unique features that distinguish it from free-living alphaproteobacteria. Using a novel fluorescence microscopy approach, we quantified R. parkeri morphology in live host cells and found that the fraction of the population undergoing cell division decreased over the course of infection. We further demonstrated the feasibility of localizing fluorescence fusions, for example, to the cell division protein ZapA, in live R. parkeri for the first time. To evaluate population growth kinetics, we developed an imaging-based assay that improves on the throughput and resolution of other methods. Finally, we applied these tools to quantitatively demonstrate that the actin homologue MreB is required for R. parkeri growth and rod shape. Collectively, a toolkit was developed of high-throughput, quantitative tools to understand growth and morphogenesis of R. parkeri that is translatable to other obligate intracellular bacteria.

Identification and Structural Modeling of the RNA Polymerase Omega Subunits in Chlamydiae and Other Obligate Intracellular Bacteria.

Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing σ factor. The core enzyme comprises two α subunits, one β subunit, one β' subunit, and one ω subunit. The ω subunit plays critical roles in the assembly of the core enzyme and other cellular functions, including the regulation of bacterial growth, the stress response, and biofilm formation. However, the identity of an ω subunit for the obligate intracellular bacterium Chlamydia has not previously been determined. Here, we report the identification of the hypothetical protein CTL0286 as the probable chlamydial ω subunit based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. Our findings indicate that CTL0286 functions as the missing ω subunit of chlamydial RNAP. Our extended analysis also indicates that all obligate intracellular bacteria have ω orthologs. IMPORTANCE Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase ω subunit, and it has been hypothesized that the chlamydial RNA polymerase ω subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle. Here, we report the identification of the chlamydial RNA polymerase ω subunit, based on conserved sequence, conserved synteny, AlphaFold-predicted conserved three-dimensional structure, and AlfaFold-Multimer-predicted conserved interactions. Our identification of the previously elusive chlamydial RNA polymerase ω subunit sets the stage for investigation of its roles in regulation of gene expression during chlamydial growth, development, and stress responses, and sets the stage for preparation and study of the intact chlamydial RNA polymerase and its interactions with inhibitors.

Orientia tsutsugamushi OtDUB Is Expressed and Interacts with Adaptor Protein Complexes during Infection.

Orientia tsutsugamushi is an etiologic agent of scrub typhus, a globally emerging rickettsiosis that can be fatal. The bacterium's obligate intracellular lifestyle requires its interaction with host eukaryotic cellular pathways. The proteins it employs to do so and their functions during infection are understudied. Recombinant versions of the recently characterized O. tsutsugamushi deubiquitylase (OtDUB) exhibit high-affinity ubiquitin binding, mediate guanine nucleotide exchange to activate Rho GTPases, bind clathrin adaptor protein complexes 1 and 2, and bind the phospholipid phosphatidylserine. Whether OtDUB is expressed and its function during O. tsutsugamushi infection have yet to be explored. Here, OtDUB expression, location, and interactome during infection were examined. O. tsutsugamushi transcriptionally and translationally expresses OtDUB throughout infection of epithelial, monocytic, and endothelial cells. Results from structured illumination microscopy, surface trypsinization of intact bacteria, and acetic acid extraction of non-integral membrane proteins indicate that OtDUB peripherally associates with the O. tsutsugamushi cell wall and is at least partially present on the bacterial surface. Analyses of the proteins with which OtDUB associates during infection revealed several known O. tsutsugamushi cell wall proteins and others. It also forms an interactome with adapter protein complex 2 and other endosomal membrane traffic regulators. This study documents the first interactors of OtDUB during O. tsutsugamushi infection and establishes a strong link between OtDUB and the host endocytic pathway.

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle.

The Rickettsiales are a group of obligate intracellular vector-borne Gram-negative bacteria that include many organisms of clinical and agricultural importance, including Anaplasma spp., Ehrlichia chaffeensis, Wolbachia, Rickettsia spp. and Orientia tsutsugamushi. This Review provides an overview of the current state of knowledge of the biology of these bacteria and their interactions with host cells, with a focus on pathogenic species or those that are otherwise important for human health. This includes a description of rickettsial genomics, bacterial cell biology, the intracellular lifestyles of Rickettsiales and the mechanisms by which they induce and evade the innate immune response.

GSMN-ML- a genome scale metabolic network reconstruction of the obligate human pathogen Mycobacterium leprae.

Leprosy, caused by Mycobacterium leprae, has plagued humanity for thousands of years and continues to cause morbidity, disability and stigmatization in two to three million people today. Although effective treatment is available, the disease incidence has remained approximately constant for decades so new approaches, such as vaccine or new drugs, are urgently needed for control. Research is however hampered by the pathogen’s obligate intracellular lifestyle and the fact that it has never been grown in vitro. Consequently, despite the availability of its complete genome sequence, fundamental questions regarding the biology of the pathogen, such as its metabolism, remain largely unexplored. In order to explore the metabolism of the leprosy bacillus with a long-term aim of developing a medium to grow the pathogen in vitro, we reconstructed an in silico genome scale metabolic model of the bacillus, GSMN-ML. The model was used to explore the growth and biomass production capabilities of the pathogen with a range of nutrient sources, such as amino acids, glucose, glycerol and metabolic intermediates. We also used the model to analyze RNA-seq data from M. leprae grown in mouse foot pads, and performed Differential Producibility Analysis to identify metabolic pathways that appear to be active during intracellular growth of the pathogen, which included pathways for central carbon metabolism, co-factor, lipids, amino acids, nucleotides and cell wall synthesis. The GSMN-ML model is thereby a useful in silico tool that can be used to explore the metabolism of the leprosy bacillus, analyze functional genomic experimental data, generate predictions of nutrients required for growth of the bacillus in vitro and identify novel drug targets.

Stem Cell-Derived Hepatocyte-Like Cells as Model for Viral Hepatitis Research.

Viral hepatitis, the leading cause of liver diseases worldwide, is induced upon infection with hepatotropic viruses, including hepatitis A, B, C, D, and E virus. Due to their obligate intracellular lifestyles, culture systems for efficient viral replication are vital. Although basic and translational research on viral hepatitis has been performed for many years, conventional hepatocellular culture systems are not optimal. These studies have greatly benefited from recent efforts on improving cell culture models for virus replication and infection studies. Here we summarize the use of human stem cell-derived hepatocyte-like cells for hepatotropic virus infection studies, including the dissection of virus-host interactions and virus-induced pathogenesis as well as the identification and validation of novel antiviral agents.

Chlamydiaceae: Diseases in Primary Hosts and Zoonosis

Bacteria of the Chlamydiaceae family are a type of Gram-negative microorganism typified by their obligate intracellular lifestyle. The majority of the members in the Chlamydiaceae family are known pathogenic organisms that primarily infect the host mucosal surfaces in both humans and animals. For instance, Chlamydia trachomatis is a well-known etiological agent for ocular and genital sexually transmitted diseases, while C. pneumoniae has been implicated in community-acquired pneumonia in humans. Other chlamydial species such as C. abortus, C. caviae, C. felis, C. muridarum, C. pecorum, and C. psittaci are important pathogens that are associated with high morbidities in animals. Importantly, some of these animal pathogens have been recognized as zoonotic agents that pose a significant infectious threat to human health through cross-over transmission. The current review provides a succinct recapitulation of the characteristics as well as transmission for the previously established members of the Chlamydiaceae family and a number of other recently described chlamydial organisms.

Ptr/CTL0175 Is Required for the Efficient Recovery of Chlamydia trachomatis From Stress Induced by Gamma-Interferon.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen in humans and a frequent cause of asymptomatic, persistent infections leading to serious complications, particularly in young women. Chlamydia displays a unique obligate intracellular lifestyle involving the infectious elementary body and the replicative reticulate body. In the presence of stressors such as gamma-interferon (IFNγ) or beta-lactam antibiotics, C. trachomatis undergoes an interruption in its replication cycle and enters a viable but non-cultivable state. Upon removal of the stressors, surviving C. trachomatis resume cell division and developmental transitions. In this report, we describe a genetic screen to identify C. trachomatis mutants with defects in recovery from IFNγ- and/or penicillin-induced stress and characterized a chemically derived C. trachomatis mutant strain that exhibited a significant decrease in recovery from IFNγ- but not penicillin-induced stress. Through lateral gene transfer and targeted insertional gene inactivation we identified ptr, encoding a predicted protease, as a gene required for recovery from IFNγ-induced stress. A C. trachomatis LGV-L2 ptr-null strain displayed reduced generation of infectious progeny and impaired genome replication upon removal of IFNγ. This defect was restored by introducing a wild type copy of ptr on a plasmid, indicating that Ptr is required for a rapid growth upon removal of IFNγ. Ptr was expressed throughout the developmental cycle and localized to the inclusion lumen. Overall, our findings indicate that the putative secreted protease Ptr is required for C. trachomatis to specifically recover from IFNγ- but not penicillin-induced stress.

Wolbachia endosymbionts subvert the endoplasmic reticulum to acquire host membranes without triggering ER stress.

The reproductive parasites Wolbachia are the most common endosymbionts on earth, present in a plethora of arthropod species. They have been introduced into mosquitos to successfully prevent the spread of vector-borne diseases, yet the strategies of host cell subversion underlying their obligate intracellular lifestyle remain to be explored in depth in order to gain insights into the mechanisms of pathogen-blocking. Like some other intracellular bacteria, Wolbachia reside in a host-derived vacuole in order to replicate and escape the immune surveillance. Using here the pathogen-blocking Wolbachia strain from Drosophila melanogaster, introduced into two different Drosophila cell lines, we show that Wolbachia subvert the endoplasmic reticulum to acquire their vacuolar membrane and colonize the host cell at high density. Wolbachia redistribute the endoplasmic reticulum, and time lapse experiments reveal tight coupled dynamics suggesting important signalling events or nutrient uptake. Wolbachia infection however does not affect the tubular or cisternal morphologies. A fraction of endoplasmic reticulum becomes clustered, allowing the endosymbionts to reside in between the endoplasmic reticulum and the Golgi apparatus, possibly modulating the traffic between these two organelles. Gene expression analyses and immunostaining studies suggest that Wolbachia achieve persistent infections at very high titers without triggering endoplasmic reticulum stress or enhanced ERAD-driven proteolysis, suggesting that amino acid salvage is achieved through modulation of other signalling pathways.

Lipid analysis of Eimeria sporozoites reveals exclusive phospholipids, a phylogenetic mosaic of endogenous synthesis, and a host-independent lifestyle

Successful inter-host transmission of most apicomplexan parasites requires the formation of infective sporozoites within the oocysts. Unlike all other infective stages that are strictly intracellular and depend on host resources, the sporozoite stage develops outside the host cells, but little is known about its self-governing metabolism. This study deployed Eimeria falciformis, a parasite infecting the mouse as its natural host, to investigate the process of phospholipid biogenesis in sporozoites. Lipidomic analyses demonstrated the occurrence of prototypical phospholipids along with abundant expression of at least two exclusive lipids, phosphatidylthreonine (PtdThr) and inositol phosphorylceramide with a phytosphingosine backbone, in sporozoites. To produce them de novo, the parasite harbors nearly the entire biogenesis network, which is an evolutionary mosaic of eukaryotic-type and prokaryotic-type enzymes. Notably, many have no phylogenetic counterpart or functional equivalent in the mammalian host. Using Toxoplasma gondii as a gene-tractable surrogate to examine Eimeria enzymes, we show a highly compartmentalized network of lipid synthesis spread primarily in the apicoplast, endoplasmic reticulum, mitochondrion, and Golgi complex. Likewise, trans-genera complementation of a Toxoplasma mutant with the PtdThr synthase from Eimeria reveals a convergent role of PtdThr in fostering the lytic cycle of coccidian parasites. Taken together, our work establishes a model of autonomous membrane biogenesis involving significant inter-organelle cooperation and lipid trafficking in sporozoites. Phylogenetic divergence of certain pathways offers attractive drug targets to block the sporulation and subsequent transmission. Not least, our results vindicate the possession of an entire de novo lipid synthesis network in a representative protist adapted to an obligate intracellular parasitic lifestyle.

Neglected zoonotic agents in cattle abortion: tackling the difficult to grow bacteria

BackgroundCoxiella burnetii, Chlamydia abortus and Leptospira spp. are difficult to grow bacteria that play a role in bovine abortion, but their diagnosis is hampered by their obligate intracellular lifestyle (C. burnetii, C. abortus) or their lability (Leptospira spp.). Their importance is based on the contagious spread in food-producing animals, but also as zoonotic agents. In Switzerland, first-line routine bacteriological diagnostics in cattle abortions is regulated by national law and includes only basic screening by staining for C. burnetii due to the high costs associated with extended spectrum analysis. The aim of this study was to assess the true occurrence of these zoonotic pathogens in 249 cases of bovine abortion in Switzerland by serology (ELISA for anti-C. burnetii and C. abortus antibodies and microscopic agglutination test for anti-Leptospira spp. antibodies), molecular methods (real-time PCR and sequencing of PCR products of Chlamydiales-positive cases), Stamp’s modification of the Ziehl-Neelsen (mod-ZN) stain and, upon availability of material, by histology and immunohistochemistry (IHC).ResultsAfter seroanalysis the prevalence was 15.9% for C. burnetii, 38.5% for C. abortus and 21.4% for Leptospira spp. By real-time PCR 12.1% and 16.9% of the cases were positive for C. burnetii and Chlamydiales, respectively, but only 2.4% were positive for C. burnetii or Chlamydiales by mod-ZN stain. Sequencing of PCR products of Chlamydiales-positive cases revealed C. abortus in 10% of cases and the presence of a mix of Chlamydiales-related bacteria in 5.2% of cases. Pathogenic Leptospira spp. were detected in 5.6% of cases. Inflammatory lesions were present histologically in all available samples which were real-time PCR-positive for Chlamydiales and Leptospira spp. One of 12 real-time PCR-positive cases for C. burnetii was devoid of histological lesions. None of the pathogens could be detected by IHC.ConclusionMolecular detection by real-time PCR complemented by histopathological analysis is recommended to improve definitive diagnosis of bovine abortion cases and determine a more accurate prevalence of these zoonotic pathogens.

Evidence for a peptidoglycan-like structure in Orientia tsutsugamushi.

Bacterial cell walls are composed of the large cross-linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan-like structure in Orientia, as well as an outer membrane containing a network of cross-linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.

Biochemical and Genetic Analysis of the Chlamydia GroEL Chaperonins.

Chaperonins are essential for cellular growth under normal and stressful conditions and consequently represent one of the most conserved and ancient protein classes. The paradigm Escherichia coli chaperonin, EcGroEL, and its cochaperonin, EcGroES, assist in the folding of proteins via an ATP-dependent mechanism. In addition to the presence of groEL and groES homologs, groEL paralogs are found in many bacteria, including pathogens, and have evolved poorly understood species-specific functions. Chlamydia spp., which are obligate intracellular bacteria, have reduced genomes that nonetheless contain three groEL genes, Chlamydia groEL (ChgroEL), ChgroEL2, and ChgroEL3 We hypothesized that ChGroEL is the bona fide chaperonin and that the paralogs perform novel Chlamydia-specific functions. To test our hypothesis, we investigated the biochemical properties of ChGroEL and its cochaperonin, ChGroES, and queried the in vivo essentiality of the three ChgroEL genes through targeted mutagenesis in Chlamydia trachomatis ChGroEL hydrolyzed ATP at a rate 25% of that of EcGroEL and bound with high affinity to ChGroES, and the ChGroEL-ChGroES complex could refold malate dehydrogenase (MDH). The chlamydial ChGroEL was selective for its cognate cochaperonin, ChGroES, while EcGroEL could function with both EcGroES and ChGroES. A P35T ChGroES mutant (ChGroESP35T) reduced ChGroEL-ChGroES interactions and MDH folding activities but was tolerated by EcGroEL. Both ChGroEL-ChGroES and EcGroEL-ChGroESP35T could complement an EcGroEL-EcGroES mutant. Finally, we successfully inactivated both paralogs but not ChgroEL, leading to minor growth defects in cell culture that were not exacerbated by heat stress. Collectively, our results support novel functions for the paralogs and solidify ChGroEL as a bona fide chaperonin that is biochemically distinct from EcGroEL.IMPORTANCEChlamydia is an important cause of human diseases, including pneumonia, sexually transmitted infections, and trachoma. The chlamydial chaperonin ChGroEL and chaperonin paralog ChGroEL2 have been associated with survival under stress conditions, and ChGroEL is linked with immunopathology elicited by chlamydial infections. However, their exact roles in bacterial survival and disease remain unclear. Our results further substantiate the hypotheses that ChGroEL is the primary chlamydial chaperonin and that the paralogs play specialized roles during infection. Furthermore, ChGroEL and the mitochondrial GroEL only functioned with their cochaperonin, in contrast to the promiscuous nature of GroEL from E. coli and Helicobacter pylori, which might indicate a divergent evolution of GroEL during the transition from a free-living organism to an obligate intracellular lifestyle.

GFPuv-Expressing Recombinant Rickettsia typhi: a Useful Tool for the Study of Pathogenesis and CD8+ T Cell Immunology in R. typhi Infection.

ABSTRACTRickettsia typhi is the causative agent of endemic typhus, a disease with increasing incidence worldwide that can be fatal. Because of its obligate intracellular life style, genetic manipulation of the pathogen is difficult. Nonetheless, in recent years, genetic manipulation tools have been successfully applied to rickettsiae. We describe here for the first time the transformation of R. typhi with the pRAM18dRGA plasmid that originally derives from Rickettsia amblyommatis and encodes the expression of GFPuv (green fluorescent protein with maximal fluorescence when excited by UV light). Transformed R. typhi (R. typhiGFPuv) bacteria are viable, replicate with kinetics similar to those of wild-type R. typhi in cell culture, and stably maintain the plasmid and GFPuv expression under antibiotic treatment in vitro and in vivo during infection of mice. CB17 SCID mice infected with R. typhiGFPuv succumb to the infection with kinetics similar to those for animals infected with wild-type R. typhi and develop comparable pathology and bacterial loads in the organs, demonstrating that the plasmid does not influence pathogenicity. In the spleen and liver of infected CB17 SCID mice, the bacteria are detectable by immunofluorescence microscopy in neutrophils and macrophages by histological staining. Finally, we show for the first time that transformed rickettsiae can be used for the detection of CD8+ T cell responses. GFP-specific restimulation of spleen cells from R. typhiGFPuv-infected BALB/c mice elicits gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin 2 (IL-2) secretion by CD8+ T cells. Thus, R. typhiGFPuv bacteria are a novel, potent tool to study infection with the pathogen in vitro and in vivo and the immune response to these bacteria.

Sugar nucleotide quantification by liquid chromatography tandem mass spectrometry reveals a distinct profile in Plasmodium falciparum sexual stage parasites.

The obligate intracellular lifestyle of Plasmodium falciparum and the difficulties in obtaining sufficient amounts of biological material have hampered the study of specific metabolic pathways in the malaria parasite. Thus, for example, the pools of sugar nucleotides required to fuel glycosylation reactions have never been studied in-depth in well-synchronized asexual parasites or in other stages of its life cycle. These metabolites are of critical importance, especially considering the renewed interest in the presence of N-, O-, and other glycans in key parasite proteins. In this work, we adapted a liquid chromatography tandem mass spectrometry (LC-MS/MS) method based on the use of porous graphitic carbon (PGC) columns and MS-friendly solvents to quantify sugar nucleotides in the malaria parasite. We report the thorough quantification of the pools of these metabolites throughout the intraerythrocytic cycle of P. falciparum. The sensitivity of the method enabled, for the first time, the targeted analysis of these glycosylation precursors in gametocytes, the parasite sexual stages that are transmissible to the mosquito vector.