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Streptococcus ruminantium is a close relative of Streptococcus suis , an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of three S. ruminantium strains isolated from bovine endocarditis in Japan.

Streptococcus parasuis is a close relative of Streptococcus suis , an important zoonotic pathogen that causes various diseases in pigs and humans. Here, we report the complete genome sequences of four strains, including the type strain of S. parasuis , isolated from the saliva of healthy pigs in Japan.

The capsule (cap) of Streptococcus suis is an anti-phagocytic element and is believed to be one of the major virulence factors of S. suis. However, we have found both cap-positive and cap-negative isolates in porcine endocarditis. The cap-negative isolates show strong adherence to platelets. Here, we compared the genome sequences of multiple cap-negative isolates with those of a cap-positive isolate from a single endocarditis case. The cap-positive and cap-negative isolates from the same pig were phylogenetically closest compared with those from other pigs. Some of the cap-negative isolates from the same pig showed different mutations in capsular polysaccharide synthesis (cps) genes, suggesting that these isolates arisen in the pig after infection. Different mutations in whole genomes were also found among isolates with identical mutations in cps genes, indicating that mutations in cps genes and the whole genome occurred independently. The calculated mutation frequencies were similar to those reported for other bacteria, suggesting all those mutations occurred spontaneously. Since cap-negative isolates are rarely found in lesions of other diseases, such as meningitis, these results suggest that endocarditis lesions may simply favored the cap-negative mutants to survive the niches, leading to their persistence in the lesions.

Background Chickens are major sources of human nutrition worldwide, but the chicken intestinal microbiota can be a source of bacterial infection. The microbiota has potential to regulate the colonization of pathogens by competitive exclusion, production of antimicrobial compounds, and stimulation of the mucosal immune system. But information on the microbiota in commercial broiler chickens is limited because of the difficulty of conducting studies at commercial farms. To obtain fundamental information that can be used to control pathogens in chickens, we determined the 6-week dynamics of microbiota in chicken cecal droppings from commercial broiler farms. Results Cecal droppings from four chickens were collected once a week from 1 to 6 weeks of age at three commercial broiler farms. A total of 168 samples were collected from 7 flocks and subjected to 16S rRNA amplicon sequencing. Despite the farms have distinctly different climate conditions, the microbiota in the same growth stages were similar among farms. Moreover, as the chickens grew and the feed types were switched, the richness and diversity of the microbiota gradually increased and convergence of the composition of the microbiota was apparent. Notably, minor bacterial taxa (i.e. OTUs with relative abundance Campylobacter. In particular, the effects of switching of feed types on the microbiota were larger than the effects of age and Campylobacter. Conclusions Irrespective of the locations of the farms, the microbiota of chicken cecum, especially minor bacteria, was successively changed more affected by feed types than by ages. Switching of feed types inducing the alteration of the microbiota may be associated with the colonization of pathogens in the chicken gut. These results will also help with extrapolation of studies in experimental animals to those in the commercial farms.

Streptococcus suis is an important zoonotic pathogen that causes major economic problems in the pig industry worldwide and serious infections in humans, including meningitis and septicemia. Here, we report the complete genome sequences of two strains isolated from asymptomatic pigs.

We report 16S rRNA amplicon sequence data from feces of 109 wild deer in Japan. The dominant bacterial taxa in fecal microbiota of wild deer hunted between village and mountainous areas and those living on Miyajima Island and in Nara Park were similar but differed in abundance.

We report 16S rRNA amplicon sequence data from feces from 58 wild boars, 60 feral raccoons, 9 wild Japanese badgers, 21 wild masked palm civets, and 8 wild raccoon dogs in Japan. The predominant bacterial taxa in the fecal microbiota were similar in part but varied among the animal species.

OBJECTIVE To evaluate colonization of Streptococcus suis and Streptococcus parasuis on pig farms in Japan and to identify sources of infections. SAMPLE Saliva, feces, and vaginal swab samples from 84 healthy pigs of several growth stages on 4 farms and swab samples of feed troughs and water dispensers at those farms. PROCEDURES Samples were collected from August 2015 to June 2016. Two quantitative PCR (qPCR) assays (one for S suis and the other for S parasuis) were designed for use in the study. The novel qPCR assays were used in combination with previously described qPCR assays for S suis serotype 2 or 1/2 and total bacteria. Relative abundance of bacteria in each sample was evaluated. RESULTS Streptococcus suis was detected in all saliva samples and some of the other samples, whereas S parasuis was not detected in any of the samples, including saliva samples, which indicated a difference in colonization preference. The ratio of S suis to total bacteria in saliva appeared to increase with age of pigs. Streptococcus suis serotype 2 or 1/2 was detected in a few saliva samples and feed trough swab samples at 2 farms where S suis infections were prevalent. CONCLUSIONS AND CLINICAL RELEVANCE Saliva, especially that of sows, appeared to be a reservoir and source of S suis infection for pigs. The qPCR assay described here may provide an effective way to monitor for S suis in live pigs, which could lead to effective disease control on pig farms.

Streptococcus parasuis has recently been removed taxonomically from Streptococcus suis, a zoonotic pathogen. S. parasuis has been detected in healthy pigs and in diseased pigs, which suggests that S. parasuis is involved in the normal microbiota of pigs and has potential pathogenicity. However, the pathogenicity of S. parasuis in pigs is unclear because of the lack of appropriate detection methods that discriminate S. parasuis from S. suis. In this study, we developed a PCR method that is specific for S. parasuis. The detection limit of the PCR was 350 CFU per reaction. Bacteria isolated from the saliva of eight pigs were collected and examined by PCR. Sixty-four isolates positive for PCR were obtained from the samples of all pigs. Thirteen of the 64 isolates were genetically confirmed as S. parasuis, and biologically and biochemically had nearly the same features of known S. parasuis strains, which suggested that strains positive for PCR were S. parasuis. Among the 64 isolates, 28 isolates were serotypes 20, 22, or 26 in the S. suis serotyping scheme. The remaining 36 isolates were untypeable, which suggested the presence of novel serotypes or a capsule-negative form. Therefore, the PCR method described in this study is a useful tool for identifying S. parasuis, and can be used in etiological studies on this bacterium.

To clarify the taxonomic classification of Streptococcus suis serotype 33, we performed biochemical and molecular genetic analyses using isolates (GUT-183, GUT-184, GUT-185, GUT-186, GUT-187T, GUT-188, GUT-189, GUT-190, GUT-191, GUT-192 and GUT-193) from bovine endocarditis. A comparative sequence analysis showed 99.2–100 % sequence similarity among the reference strain of S. suis serotype 33 and our isolates for the 16S rRNA gene. These similarities were higher than those between the isolate GUT-187T and S. suis and other streptococci. Comparison of sodA genes also showed high degrees of similarities among the reference strain of S. suis serotype 33 and our isolates (99.7–100 %), which were higher than those between the GUT-187T and S. suis and other streptococci. DNA–DNA relatedness among three isolates (GUT-186, GUT-187T, the reference strain of S. suis serotype 33) was over 76.7 %. In contrast, the relatedness between GUT-187T and the other streptococcal species ( S. suis , Streptococcus parasuis , Streptococcus acidominimus and Streptococcus porci ) was 8.4–24.9 %. Phylogenetic analyses showed that the isolates did not affiliate closely to any known species of the genus Streptococcus . Moreover, GUT-187T could be distinguished from S. suis and other closely related species of genus Streptococcus using biochemical tests. On the basis of the phenotypic and molecular genetic data, we propose that the isolates of S. suis serotype 33 should be classified into the genus Streptococcus , Streptococcus ruminantium sp. nov. with the type strain GUT-187T (=DSM 104980T=JCM 31869T).

Streptococcus parasuis has recently been removed taxonomically from Streptococcus suis, a zoonotic pathogen. S. parasuis has been detected in healthy pigs and in diseased pigs, which suggests that S. parasuis is involved in the normal microbiota of pigs and has potential pathogenicity. However, the pathogenicity of S. parasuis in pigs is unclear because of the lack of appropriate detection methods that discriminate S. parasuis from S. suis. In this study, we developed a PCR method that is specific for S. parasuis. The detection limit of the PCR was 350 CFU per reaction. Bacteria isolated from the saliva of eight pigs were collected and examined by PCR. Sixty-four isolates positive for PCR were obtained from the samples of all pigs. Thirteen of the 64 isolates were genetically confirmed as S. parasuis, and biologically and biochemically had nearly the same features of known S. parasuis strains, which suggested that strains positive for PCR were S. parasuis. Among the 64 isolates, 28 isolates were serotypes 20, 22, or 26 in the S. suis serotyping scheme. The remaining 36 isolates were untypeable, which suggested the presence of novel serotypes or a capsule-negative form. Therefore, the PCR method described in this study is a useful tool for identifying S. parasuis, and can be used in etiological studies on this bacterium.

We have previously demonstrated conjugation of Escherichia coli into vacuoles of the protozoal ciliate (Tetrahymena thermophila). This indicated a possible role of ciliates in evoking bacterial quorum sensing, directly connecting bacterial survival via accumulation in the ciliate vacuoles. We therefore assessed if ciliates promoted bacterial autoinducer (AI)-2 accumulation with vacuole formation, which controls quorum sensing. E. coli AI-2 accumulation was significantly enhanced in the supernatants of a mixed culture of ciliates and bacteria, likely depending on ciliate density rather than bacterial concentration. As expected, AI-2 production was significantly correlated with vacuole formation. The experiment with E. coli luxS mutants showed that ciliates failed to enhance bacterial AI-2 accumulation, denying a nonspecific phenomenon. Fluorescence microscopy revealed accumulation of fragmented bacteria in ciliate vacuoles, and, more importantly, expulsion of the vacuoles containing disrupted bacteria into the culture supernatant. There was no increase in the expression of luxS (encoding AI-2) or ydgG (a transporter for controlling bacterial export of AI-2). We conclude that ciliates promote bacterial AI-2 accumulation in a mixed culture, via accumulation of disrupted bacteria in ciliate vacuoles followed by expulsion of the vacuoles, independently of luxS or ydgG gene induction. This is believed to be the first demonstration of a relationship between E. coli AI-2 dynamics and ciliates. In the natural environment, ciliate biotopes may provide a survival advantage to bacteria inhabiting such biotopes, via evoking quorum sensing.

It is generally difficult to specify the sources of infection by which domestic animals may acquire pathogens. Through 16S rRNA gene amplicon sequencing, we compared the composition of microbiota in the saliva, vaginal mucus, and feces of pigs, and in swabs of feeder troughs and water dispensers collected from pig farms in Vietnam. The composition of the microbiota differed between samples in each sample group. Streptococcus, Actinobacillus, Moraxella, and Rothia were the most abundant genera and significantly discriminative in saliva samples, regardless of the plasticity and changeability of the composition of microbiota in saliva. Moreover, species assignment of the genus Streptococcus revealed that Streptococcus suis was exceptional in the salivary microbiota, due to being most abundant among the streptococcal species and sharing estimated proportions of 5.7%-9.4% of the total bacteria in saliva. Thus, pig oral microbiota showed unique characteristics in which the major species was the pig pathogen. On the other hand, β-diversity analysis showed that the microbiota in saliva was distinct from those in the others. From the above results, pig saliva was shown to be the major natural habitat of S. suis, and is suggested to be the most probable source of S. suis infection.

Summary To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

We have previously demonstrated conjugation of Escherichia coli into vacuoles of the protozoal ciliate (Tetrahymena thermophila). This indicated a possible role of ciliates in evoking bacterial quorum sensing, directly connecting bacterial survival via accumulation in the ciliate vacuoles. We therefore assessed if ciliates promoted bacterial autoinducer (AI)-2 accumulation with vacuole formation, which controls quorum sensing. E. coli AI-2 accumulation was significantly enhanced in the supernatants of a mixed culture of ciliates and bacteria, likely depending on ciliate density rather than bacterial concentration. As expected, AI-2 production was significantly correlated with vacuole formation. The experiment with E. coli luxS mutants showed that ciliates failed to enhance bacterial AI-2 accumulation, denying a nonspecific phenomenon. Fluorescence microscopy revealed accumulation of fragmented bacteria in ciliate vacuoles, and, more importantly, expulsion of the vacuoles containing disrupted bacteria into the culture supernatant. There was no increase in the expression of luxS (encoding AI-2) or ydgG (a transporter for controlling bacterial export of AI-2). We conclude that ciliates promote bacterial AI-2 accumulation in a mixed culture, via accumulation of disrupted bacteria in ciliate vacuoles followed by expulsion of the vacuoles, independently of luxS or ydgG gene induction. This is believed to be the first demonstration of a relationship between E. coli AI-2 dynamics and ciliates. In the natural environment, ciliate biotopes may provide a survival advantage to bacteria inhabiting such biotopes, via evoking quorum sensing.

The obligate intracellular bacterium Chlamydia pneumoniae is not only a causative agent of community-acquired pneumonia but is also associated with a more serious chronic disease, asthma, which might be exacerbated by air pollution containing carbon nanoparticles. Although a detailed mechanism of exacerbation remains unknown, the proinflammatory cytokine interleukin-1β (IL-1β) is a critical player in the pathogenesis of asthma. C. pneumoniae induces IL-1β in macrophages via NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activation and Toll-like receptor 2/4 (TLR2/4) stimulation. Carbon nanoparticles, such as carbon nanotubes (CNTs), can also evoke the NLRP3 inflammasome to trigger IL-1β secretion from lipopolysaccharide-primed macrophages. This study assessed whether costimulation of C. pneumoniae with CNTs synergistically enhanced IL-1β secretion from macrophages, and determined the molecular mechanism involved. Enhanced IL-1β secretion from C. pneumoniae -infected macrophages by CNTs was dose and time dependent. Transmission electron microscopy revealed that C. pneumoniae and CNTs were engulfed concurrently by macrophages. Inhibitors of actin polymerization or caspase-1, a component of the inflammasome, significantly blocked IL-1β secretion. Gene silencing using small interfering RNA (siRNA) targeting the NLRP3 gene also abolished IL-1β secretion. Other inhibitors (K + efflux inhibitor, cathepsin B inhibitor, and reactive oxygen species-generating inhibitor) also blocked IL-1β secretion. Taken together, these findings demonstrated that CNTs synergistically enhanced IL-1β secretion from C. pneumoniae -infected macrophages via the NLRP3 inflammasome and caspase-1 activation, providing novel insight into our understanding of how C. pneumoniae infection can exacerbate asthma.

Here, we report the draft genome sequence of high-temperature-adapted Protochlamydia sp. strain HS-T3, an environmental chlamydia. This bacterium is an amoebal endosymbiont, found in Acanthamoeba isolated from a hot spring in Japan. Strain HS-T3 readily grew in mammalian cells at 37°C, a characteristic not previously reported for environmental chlamydiae.

To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

Obligate amoebal endosymbiotic bacterium Protochlamydia with ancestral pathogenic chlamydial features evolved to survive within protist hosts, such as Acanthamoba, 0.7-1.4 billion years ago, but not within vertebrates including humans. This observation raises the possibility that interactions between Protochlamydia and human cells may result in a novel cytopathic effect, leading to new insights into host-parasite relationships. Previously, we reported that Protochlamydia induces apoptosis of the immortalized human cell line, HEp-2. In this study, we attempted to elucidate the molecular mechanism underlying this apoptosis. We first confirmed that, upon stimulation with the bacteria, poly (ADP-ribose) polymerase (PARP) was cleaved at an early stage in HEp-2 cells, which was dependent on the amount of bacteria. A pan-caspase inhibitor and both caspase-3 and -9 inhibitors similarly inhibited the apoptosis of HEp-2 cells. A decrease of the mitochondrial membrane potential was also confirmed. Furthermore, lactacystin, an inhibitor of chlamydial protease-like activity factor (CPAF), blocked the apoptosis. Cytochalasin D also inhibited the apoptosis, which was dependent on the drug concentration, indicating that bacterial entry into cells was required to induce apoptosis. Interestingly, Yersinia type III inhibitors (ME0052, ME0053, and ME0054) did not have any effect on the apoptosis. We also confirmed that the Protochlamydia used in this study possessed a homologue of the cpaf gene and that two critical residues, histidine-101 and serine-499 of C. trachomatis CPAF in the active center, were conserved. Thus, our results indicate that after entry, Protochlamydia-secreted CPAF induces mitochondrial dysfunction with a decrease of the membrane potential, followed by caspase-9, caspase-3 and PARP cleavages for apoptosis. More interestingly, because C. trachomatis infection can block the apoptosis, our finding implies unique features of CPAF between pathogenic and primitive chlamydiae.

Lymphocytes are a potential host cell for Chlamydophila pneumoniae, although why the bacteria must hide in lymphocytes remains unknown. Meanwhile, interferon (IFN)-γ is a crucial factor for eliminating chlamydiae from infected cells through indoleamine 2,3-dioxygenase (IDO) expression, resulting in depletion of tryptophan. We therefore assessed if lymphocytes could work as a shelter for the bacteria to escape IFN-γ. C. pneumoniae grew normally in human lymphoid Jurkat cells, even in the presence of IFN-γ or under stimulation with phorbol myristate acetate plus ionomycin. Although Jurkat cells expressed IFN-γ receptor CD119, their lack of IDO expression was confirmed by RT-PCR and western blotting. Also, C. pneumoniae survived in enriched human peripheral blood lymphocytes, even in the presence of IFN-γ. Furthermore, C. pneumoniae in spleen cells obtained from IFN-γ knockout mice with C57BL/6 background was maintained in a similar way to wild-type mice, supporting a minimal role of IFN-γ-related response for eliminating C. pneumoniae from lymphocytes. Thus, we concluded that IFN-γ did not remove C. pneumoniae from lymphocytes, possibly providing a shelter for C. pneumoniae to escape from the innate immune response, which has direct clinical significance.

Chlamydia trachomatis L2 invasively attacks lymphatic and subepithelial tissues of the genital tract during the formation of primary lesions. This subsequently results in lymphadenopathy, and suggests a greater propensity for systemic dissemination. However, whether lymphocytes are a potential vehicle cell for the dissemination of this infection remains unknown. We therefore assessed the growth properties of C. trachomatis L2 in lymphoid Jurkat cells compared with those observed in epithelial HeLa cells. Both cells supported the growth of C. trachomatis with a similar increase in infective progenies. Enriched human-blood lymphocytes also supported the C. trachomatis growth as well as Jurkat cells. Bacteria infecting the Jurkat cells were more susceptible to antibiotics (doxycycline, azithromycin, ofloxacin) than those in HeLa cells. Of the sphingomyelin biosynthesis inhibitors tested, both myriocin and fumonisin B1 significantly inhibited bacterial growth in both cells types. A Jurkat cell mutant that impaired bacterial growth was established using ethylmethanesulfonate treatment. DNA microarray analysis with real-time reverse transcription-polymerase chain reaction revealed that the mutant cells over-expressed granzyme K gene. Immunofluorescence staining also indicated that granzyme K irregularly over-expressed among the mutant cells as compared with that of the wild cells, suggesting a possible mechanism refractory to C. trachomatis infection. Thus, we concluded that C. trachomatis L2 could infect Jurkat cells with lymphoid properties, providing a new tool for studying C. trachomatis dissemination to tissues via lymphocyte movement.

We have previously demonstrated that the steroid receptor antagonist mifepristone (RU486) causes growth inhibition of Chlamydophila pneumoniae by binding to and subsequently destroying the bacteria during their normal developmental cycle in epithelial HEp-2 cells. In the present study, we assessed the efficacy of treatment with RU486 against persistent C. pneumoniae infection in interferon (IFN)γ-treated HEp-2 cells. Assessment of bacterial growth modification, the number of infectious progenies, the formation of inclusions, and the expressions of the C. pneumoniae genes 16S rRNA and hsp60 were investigated in cells with or without IFNγ stimulation in the presence of RU486, using an inclusion-forming unit (IFU) assay, fluorescence microscopic analysis, and reverse transcription polymerase chain reaction (RT-PCR), respectively. Our results indicated that RU486 treatment produced growth inhibition and an absence of C. pneumoniae gene expression in normal HEp-2 cells and that this treatment failed to inhibit C. pneumoniae growth in HEp-2 cells stimulated with IFNγ. These results indicate that treatment with RU486 had a limited effect on C. pneumoniae growth only during the active developmental stage of the bacteria, suggesting that the bacterial target molecule of RU486 is not expressed sufficiently during persistent infection in which there is an aberrant developmental cycle. Thus, our findings provide valuable insight into the complicated chlamydial biological processes involved in the recurrent cycling between normal and persistent infections.

Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7-1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37 °C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30 °C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30 °C compared to at 37 °C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.

The authors describe a rule-based logic reorganization system LORES/EX, which transforms an existing logic circuit into one that is dependent on other technology. The system can be not only flexibly adjustable to technology changes, but also has several special features as follows: (1) introduction of the circuit standardization rules makes the size of the rule base much smaller, and makes the description and modification of the rules easier; (2) application of conflict resolution based on evaluation functions contributes to the generation of high-quality circuits; and (3) introduction of the automatic partitioning of a circuit into subcircuits enables the reorganization of the circuit with some 10 K gates in a practical computation time. >

Background: Pathogenic chlamydiae are obligate intracellular pathogens and have adapted successfully to human cells, causing sexually transmitted diseases or pneumonia. Chlamydial outer protein N (CopN) is likely a critical effector protein secreted by the type III secretion system in chlamydiae, which manipulates host cells. However, the mechanisms of its action remain to be clarified. In this work, we aimed to identify previously unidentified CopN effector target in host cells. Results: We first performed a pull-down assay with recombinant glutathione S-transferase (GST) fusion CopN proteins (GST-CpCopN: Chlamydia pneumoniae TW183, GST-CtCopN: Chlamydia trachomatis D/UW-3/CX) as "bait" and soluble lysates obtained from human immortal epithelial HEp-2 cells as "prey", followed by SDS-PAGE with mass spectroscopy (MS). We found that a host cell protein specifically bound to GST-CpCopN, but not GST-CtCopN. MS revealed the host protein to be fructose bisphosphate aldolase A (aldolase A), which plays a key role in glycolytic metabolism. We also confirmed the role of aldolase A in chlamydia-infected HEp-2 cells by using two distinct experiments for gene knockdown with an siRNA specific to aldolase A transcripts, and for assessment of glycolytic enzyme gene expression levels. As a result, both the numbers of chlamydial inclusion-forming units and RpoD transcripts were increased in the chlamydia-infected aldolase A knockdown cells, as compared with the wild-type HEp-2 cells. Meanwhile, chlamydial infection tended to enhance expression of aldolase A. Conclusions: We discovered that one of the C. pneumoniae CopN targets is the glycolytic enzyme aldolase A. Sequestering aldolase A may be beneficial to bacterial growth in infected host cells.

Previous work has shown that the obligate intracellular amoebal endosymbiont Neochlamydia S13, an environmental chlamydia strain, has an amoebal infection rate of 100%, but does not cause amoebal lysis and lacks transferability to other host amoebae. The underlying mechanism for these observations remains unknown. In this study, we found that the host amoeba could completely evade Legionella infection. The draft genome sequence of Neochlamydia S13 revealed several defects in essential metabolic pathways, as well as unique molecules with leucine-rich repeats (LRRs) and ankyrin domains, responsible for protein-protein interaction. Neochlamydia S13 lacked an intact tricarboxylic acid cycle and had an incomplete respiratory chain. ADP/ATP translocases, ATP-binding cassette transporters, and secretion systems (types II and III) were well conserved, but no type IV secretion system was found. The number of outer membrane proteins (OmcB, PomS, 76-kDa protein, and OmpW) was limited. Interestingly, genes predicting unique proteins with LRRs (30 genes) or ankyrin domains (one gene) were identified. Furthermore, 33 transposases were found, possibly explaining the drastic genome modification. Taken together, the genomic features of Neochlamydia S13 explain the intimate interaction with the host amoeba to compensate for bacterial metabolic defects, and illuminate the role of the endosymbiont in the defense of the host amoebae against Legionella infection.