Scrutinizing the immune defence inventory of Camponotus floridanus applying total transcriptome sequencing.

BACKGROUND: Defence mechanisms of organisms are shaped by their lifestyle, environment and pathogen pressure. Carpenter ants are social insects which live in huge colonies comprising genetically closely related individuals in high densities within nests. This lifestyle potentially facilitates the rapid spread of pathogens between individuals. In concert with their innate immune system, social insects may apply external immune defences to manipulate the microbial community among individuals and within nests. Additionally, carpenter ants carry a mutualistic intracellular and obligate endosymbiotic bacterium, possibly maintained and regulated by the innate immune system. Thus, different selective forces could shape internal immune defences of Camponotus floridanus. RESULTS: The immune gene repertoire of C. floridanus was investigated by re-evaluating its genome sequence combined with a full transcriptome analysis of immune challenged and control animals using Illumina sequencing. The genome was re-annotated by mapping transcriptome reads and masking repeats. A total of 978 protein sequences were characterised further by annotating functional domains, leading to a change in their original annotation regarding function and domain composition in about 8% of all proteins. Based on homology analysis with key components of major immune pathways of insects, the C. floridanus immune-related genes were compared to those of Drosophila melanogaster, Apis mellifera, and other hymenoptera. This analysis revealed that overall the immune system of carpenter ants comprises many components found in these insects. In addition, several C. floridanus specific genes of yet unknown functions but which are strongly induced after immune challenge were discovered. In contrast to solitary insects like Drosophila or the hymenopteran Nasonia vitripennis, the number of genes encoding pattern recognition receptors specific for bacterial peptidoglycan (PGN) and a variety of known antimicrobial peptide (AMP) genes is lower in C. floridanus. The comparative analysis of gene expression post immune-challenge in different developmental stages of C. floridanus suggests a stronger induction of immune gene expression in larvae in comparison to adults. CONCLUSIONS: The comparison of the immune system of C. floridanus with that of other insects revealed the presence of a broad immune repertoire. However, the relatively low number of PGN recognition proteins and AMPs, the identification of Camponotus specific putative immune genes, and stage specific differences in immune gene regulation reflects Camponotus specific evolution including adaptations to its lifestyle.

Transcriptional profiling of Bordetella pertussis reveals requirement of RNA chaperone Hfq for Type III secretion system functionality.

Bordetella pertussis, the causative agent of human whooping cough (pertussis) produces a complex array of virulence factors in order to establish efficient infection in the host. The RNA chaperone Hfq and small regulatory RNAs are key players in posttranscriptional regulation in bacteria and have been shown to play an essential role in virulence of a broad spectrum of bacterial pathogens. This study represents the first attempt to characterize the Hfq regulon of the human pathogen B. pertussis under laboratory conditions as well as upon passage in the host and indicates that loss of Hfq has a profound effect on gene expression in B. pertussis. Comparative transcriptional profiling revealed that Hfq is required for expression of several virulence factors in B. pertussis cells including the Type III secretion system (T3SS). In striking contrast to the wt strain, T3SS did not become operational in the hfq mutant passaged either through mice or macrophages thereby proving that Hfq is required for the functionality of the B. pertussis T3SS. Likewise, expression of virulence factors vag8 and tcfA encoding autotransporter and tracheal colonization factor, respectively, was strongly reduced in the hfq mutant. Importantly, for the first time we demonstrate that B. pertussis T3SS can be activated upon contact with macrophage cells in vitro.

Development and characterization of attenuated metabolic mutants of Bordetella bronchiseptica for applications in vaccinology.

Bordetella bronchiseptica is an important pathogen causing a number of veterinary respiratory syndromes in agriculturally important and food-producing confinement-reared animals, resulting in great economic losses annually amounting to billions of euros worldwide. Currently available live vaccines are incompletely satisfactory in terms of efficacy and safety. An efficient vaccine for livestock animals would allow reducing the application of antibiotics, thereby preventing the massive release of pharmaceuticals into the environment. Here, we describe two new potential vaccine strains based on the BB7865 strain. Two independent attenuating mutations were incorporated by homologous recombination in order to make negligible the risk of recombination and subsequent reversion to the virulent phenotype. The mutations are critical for bacterial metabolism, resistance to oxidative stress, intracellular survival and in vivo persistence. The resulting double mutants BB7865 risA aroA and BB7865 risA dapE were characterized as promising vaccine candidates, which are able to confer protection against colonization of the lower respiratory tract after sublethal challenge with the wild-type strain.

Immunity and symbiosis.

The invertebrate immune system, which has become a major research focus, shares basic features of innate immunity with vertebrates and men. A special feature apparently found only in invertebrates is their close association with vertically heritable symbiotic microorganisms. The validity of the simple view of symbiosis as a mutually beneficial interaction between two uneven partners mainly improving the nutritional state of the two companions has been challenged, however, as symbiotic interactions might involve more partners, and symbiotic functions of the microorganisms are much more diverse than previously assumed. Likewise, microorganisms considered to be mostly harmful to their hosts have been shown to enhance host fitness under some circumstances. The role of a symbiont itself might change between environments or life stages of the host and symbionts might have features previously thought to be specific for pathogens. Understanding symbiotic interactions requires the comprehension of the cross-talk between the symbiotic companions, and the dissection of how long-lasting infections are established without eliminating the symbiont by host immune responses. Fascinating new findings in this field revealed that symbiosis might contribute to defence against pathogens or natural enemies. New symbiont-based approaches to defeat agricultural pests or pathogen transmission by arthropod vectors are becoming conceivable.

Bacteriocyte dynamics during development of a holometabolous insect, the carpenter ant Camponotus floridanus.

BACKGROUND: The carpenter ant Camponotus floridanus harbors obligate intracellular mutualistic bacteria (Blochmannia floridanus) in specialized cells, the bacteriocytes, intercalated in their midgut tissue. The diffuse distribution of bacteriocytes over the midgut tissue is in contrast to many other insects carrying endosymbionts in specialized tissues which are often connected to the midgut but form a distinct organ, the bacteriome. C. floridanus is a holometabolous insect which undergoes a complete metamorphosis. During pupal stages a complete restructuring of the inner organs including the digestive tract takes place. So far, nothing was known about maintenance of endosymbionts during this life stage of a holometabolous insect. It was shown previously that the number of Blochmannia increases strongly during metamorphosis. This implicates an important function of Blochmannia in this developmental phase during which the animals are metabolically very active but do not have access to external food resources. Previous experiments have shown a nutritional contribution of the bacteria to host metabolism by production of essential amino acids and urease-mediated nitrogen recycling. In adult hosts the symbiosis appears to degenerate with increasing age of the animals. RESULTS: We investigated the distribution and dynamics of endosymbiotic bacteria and bacteriocytes at different stages during development of the animals from larva to imago by confocal laser scanning microscopy. The number of bacteriocytes in relation to symbiont-free midgut cells varied strongly over different developmental stages. Especially during metamorphosis the relative number of bacteria-filled bacteriocytes increased strongly when the larval midgut epithelium is shed. During this developmental stage the midgut itself became a huge symbiotic organ consisting almost exclusively of cells harboring bacteria. In fact, during this phase some bacteria were also found in midgut cells other than bacteriocytes indicating a cell-invasive capacity of Blochmannia. In adult animals the number of bacteriocytes generally decreased. CONCLUSIONS: During the life cycle of the animals the distribution of bacteriocytes and of Blochmannia endosymbionts is remarkably dynamic. Our data show how the endosymbiont is retained within the midgut tissue during metamorphosis thereby ensuring the maintenance of the intracellular endosymbiosis despite a massive reorganization of the midgut tissue. The transformation of the entire midgut into a symbiotic organ during pupal stages underscores the important role of Blochmannia for its host in particular during metamorphosis.

Resemblance and divergence: the "new" members of the genus Bordetella.

Bordetella pertussis, the etiological agent of whooping cough, belongs to the bacterial pathogens first described in the so-called golden era of microbiology more than 100 years ago. In the course of the following decades, several other closely related pathogens were described which are nowadays classified in the genus Bordetella together with B. pertussis. These are the human and animal pathogens B. parapertussis, B. bronchiseptica and B. avium which are of high medical or veterinary interest, and which, together with B. pertussis, are referred to as the "classical" Bordetella species. Only in the past 15 years, several additional species were classified in the genus, frequently isolated from patients with underlying disease, animals or from the environment. Very little is known about most of these bacteria. In the present review, the current knowledge about these "new" Bordetella species is briefly summarized.

Formicine ants swallow their highly acidic poison for gut microbial selection and control.

Animals continuously encounter microorganisms that are essential for health or cause disease. They are thus challenged to control harmful microbes while allowing the acquisition of beneficial microbes. This challenge is likely especially important for social insects with respect to microbes in food, as they often store food and exchange food among colony members. Here we show that formicine ants actively swallow their antimicrobial, highly acidic poison gland secretion. The ensuing acidic environment in the stomach, the crop, can limit the establishment of pathogenic and opportunistic microbes ingested with food and improve the survival of ants when faced with pathogen contaminated food. At the same time, crop acidity selectively allows acquisition and colonization by Acetobacteraceae, known bacterial gut associates of formicine ants. This suggests that swallowing of the poison in formicine ants acts as a microbial filter and that antimicrobials have a potentially widespread but so far underappreciated dual role in host-microbe interactions.

Activity of Tracheal Cytotoxin of Bordetella pertussis in a Human Tracheobronchial 3D Tissue Model.

Bordetella pertussis is a highly contagious pathogen which causes whooping cough in humans. A major pathophysiology of infection is the extrusion of ciliated cells and subsequent disruption of the respiratory mucosa. Tracheal cytotoxin (TCT) is the only virulence factor produced by B. pertussis that has been able to recapitulate this pathology in animal models. This pathophysiology is well characterized in a hamster tracheal model, but human data are lacking due to scarcity of donor material. We assessed the impact of TCT and lipopolysaccharide (LPS) on the functional integrity of the human airway mucosa by using in vitro airway mucosa models developed by co-culturing human tracheobronchial epithelial cells and human tracheobronchial fibroblasts on porcine small intestinal submucosa scaffold under airlift conditions. TCT and LPS either alone and in combination induced blebbing and necrosis of the ciliated epithelia. TCT and LPS induced loss of ciliated epithelial cells and hyper-mucus production which interfered with mucociliary clearance. In addition, the toxins had a disruptive effect on the tight junction organization, significantly reduced transepithelial electrical resistance and increased FITC-Dextran permeability after toxin incubation. In summary, the results indicate that TCT collaborates with LPS to induce the disruption of the human airway mucosa as reported for the hamster tracheal model.

Promoter characterization in the AT-rich genome of the obligate endosymbiont "Candidatus Blochmannia floridanus".

The characterization of transcriptional start sites of 14 genes encoded by the extremely AT-rich genome of "Candidatus Blochmannia floridanus" revealed a high degree of conservation with the RpoD promoter consensus sequence of the free-living relative Escherichia coli. Moreover, in agreement with the presence of the alternative heat shock sigma factor RpoH in "Ca. Blochmannia," typical RpoH-dependent promoters were identified. However, no heat shock response resembling that of E. coli could be detected in "Ca. Blochmannia."

JANE: efficient mapping of prokaryotic ESTs and variable length sequence reads on related template genomes.

BACKGROUND: ESTs or variable sequence reads can be available in prokaryotic studies well before a complete genome is known. Use cases include (i) transcriptome studies or (ii) single cell sequencing of bacteria. Without suitable software their further analysis and mapping would have to await finalization of the corresponding genome. RESULTS: The tool JANE rapidly maps ESTs or variable sequence reads in prokaryotic sequencing and transcriptome efforts to related template genomes. It provides an easy-to-use graphics interface for information retrieval and a toolkit for EST or nucleotide sequence function prediction. Furthermore, we developed for rapid mapping an enhanced sequence alignment algorithm which reassembles and evaluates high scoring pairs provided from the BLAST algorithm. Rapid assembly on and replacement of the template genome by sequence reads or mapped ESTs is achieved. This is illustrated (i) by data from Staphylococci as well as from a Blattabacteria sequencing effort, (ii) mapping single cell sequencing reads is shown for poribacteria to sister phylum representative Rhodopirellula Baltica SH1. The algorithm has been implemented in a web-server accessible at http://jane.bioapps.biozentrum.uni-wuerzburg.de. CONCLUSION: Rapid prokaryotic EST mapping or mapping of sequence reads is achieved applying JANE even without knowing the cognate genome sequence.

Transcriptional profiling of the endosymbiont Blochmannia floridanus during different developmental stages of its holometabolous ant host.

The transcriptome of Blochmannia floridanus, the endosymbiont of the carpenter ant Camponotus floridanus, is presented during various developmental stages of its holometabolous host by use of a whole-genome DNA macroarray. The detected transcription patterns indicate the presence of local transcription units as well as global regulatory mechanisms. Yet, the overall regulation scale is very modest, rarely exceeding a factor of three. A large number of genes show differential expression in different life stages and a distinct expression pattern of genes possibly involved in symbiotic function as compared with housekeeping genes is apparent. However, these transcriptional changes are small as compared with the changes in the number of bacteria during host development, which is the highest in pupae and in young imagines. Control of replication of the bacteria in certain life stages may therefore be the decisive parameter influencing the overall level of gene expression of Blochmannia in the animal. The few highly expressed genes like those encoding molecular chaperones exhibit a significantly higher G+C content than moderately expressed genes.

Insects as hosts for mutualistic bacteria.

Insects are among the most successful animals on Earth both with regard to their biomass and biodiversity. It is estimated that up to 20% of all insects are obligately associated with symbiotic microorganisms, and it is likely that their capacity to engage microbial companions has greatly contributed to their evolutionary success. The main focus of this review lies on obligately intracellular bacteria residing in specialized cells, the bacteriocytes, provided by the host. In the past few years the focus in research on these bacteria has been on their biological role for the host and the consequences on the genome and metabolic capacities shaped by a long-lasting obligate association confined to the interior of a eukaryotic host cell. Here, we compare those endosymbiont-host interactions where the genome of the bacterium is sequenced.

Genomic island excisions in Bordetella petrii.

BACKGROUND: Among the members of the genus Bordetella B. petrii is unique, since it is the only species isolated from the environment, while the pathogenic Bordetellae are obligately associated with host organisms. Another feature distinguishing B. petrii from the other sequenced Bordetellae is the presence of a large number of mobile genetic elements including several large genomic regions with typical characteristics of genomic islands collectively known as integrative and conjugative elements (ICEs). These elements mainly encode accessory metabolic factors enabling this bacterium to grow on a large repertoire of aromatic compounds. RESULTS: During in vitro culture of Bordetella petrii colony variants appear frequently. We show that this variability can be attributed to the presence of a large number of metastable mobile genetic elements on its chromosome. In fact, the genome sequence of B. petrii revealed the presence of at least seven large genomic islands mostly encoding accessory metabolic functions involved in the degradation of aromatic compounds and detoxification of heavy metals. Four of these islands (termed GI1 to GI3 and GI6) are highly related to ICEclc of Pseudomonas knackmussii sp. strain B13. Here we present first data about the molecular characterization of these islands. We defined the exact borders of each island and we show that during standard culture of the bacteria these islands get excised from the chromosome. For all but one of these islands (GI5) we could detect circular intermediates. For the clc-like elements GI1 to GI3 of B. petrii we provide evidence that tandem insertion of these islands which all encode highly related integrases and attachment sites may also lead to incorporation of genomic DNA which originally was not part of the island and to the formation of huge composite islands. By integration of a tetracycline resistance cassette into GI3 we found this island to be rather unstable and to be lost from the bacterial population within about 100 consecutive generations. Furthermore, we show that GI3 is self transmissible and by conjugation can be transferred to B. bronchiseptica thus proving it to be an active integrative and conjugative element CONCLUSION: The results show that phenotypic variation of B. petrii is correlated with the presence of genomic islands. Tandem integration of related islands may contribute to island evolution by the acquisition of genes originally belonging to the bacterial core genome. In conclusion, B. petrii appears to be the first member of the genus in which horizontal gene transfer events have massively shaped its genome structure.

Metabolic interdependence of obligate intracellular bacteria and their insect hosts.

Mutualistic associations of obligate intracellular bacteria and insects have attracted much interest in the past few years due to the evolutionary consequences for their genome structure. However, much less attention has been paid to the metabolic ramifications for these endosymbiotic microorganisms, which have to compete with but also to adapt to another metabolism--that of the host cell. This review attempts to provide insights into the complex physiological interactions and the evolution of metabolic pathways of several mutualistic bacteria of aphids, ants, and tsetse flies and their insect hosts.

The missing link: Bordetella petrii is endowed with both the metabolic versatility of environmental bacteria and virulence traits of pathogenic Bordetellae.

BACKGROUND: Bordetella petrii is the only environmental species hitherto found among the otherwise host-restricted and pathogenic members of the genus Bordetella. Phylogenetically, it connects the pathogenic Bordetellae and environmental bacteria of the genera Achromobacter and Alcaligenes, which are opportunistic pathogens. B. petrii strains have been isolated from very different environmental niches, including river sediment, polluted soil, marine sponges and a grass root. Recently, clinical isolates associated with bone degenerative disease or cystic fibrosis have also been described. RESULTS: In this manuscript we present the results of the analysis of the completely annotated genome sequence of the B. petrii strain DSMZ12804. B. petrii has a mosaic genome of 5,287,950 bp harboring numerous mobile genetic elements, including seven large genomic islands. Four of them are highly related to the clc element of Pseudomonas knackmussii B13, which encodes genes involved in the degradation of aromatics. Though being an environmental isolate, the sequenced B. petrii strain also encodes proteins related to virulence factors of the pathogenic Bordetellae, including the filamentous hemagglutinin, which is a major colonization factor of B. pertussis, and the master virulence regulator BvgAS. However, it lacks all known toxins of the pathogenic Bordetellae. CONCLUSION: The genomic analysis suggests that B. petrii represents an evolutionary link between free-living environmental bacteria and the host-restricted obligate pathogenic Bordetellae. Its remarkable metabolic versatility may enable B. petrii to thrive in very different ecological niches.

Immune reactions of insects on bacterial pathogens and mutualists.

In the past few years the knowledge of insect defense mechanisms against pathogenic microorganisms and parasites has significantly increased on both the molecular and the organismic level. These investigations have led to new concepts of immune protection also relevant for mammals with the identification of the Toll receptor family as an eminent example. This review provides a brief overview of insect strategies to on the one hand defeat bacterial pathogens while on the other hand cooperating with symbiotic bacteria beneficial for the insects.

Regulation of bacterial virulence by two-component systems.

In bacteria, two-component systems (TCS) are widely used signal transduction devices which are engaged in a multitude of gene regulatory systems that respond to changing growth conditions. Many pathogenic bacteria encounter different microenvironments during their infectious cycle and their ability to efficiently adapt to different niches inside and outside of their host organisms is frequently mediated by TCSs, which can, therefore, be considered as an essential prerequisite for their pathogenicity. Although significant progress has been made in the elucidation of basic principles of the signal transduction process itself, in many pathogens the contribution of TCS to bacterial virulence is insufficiently recognized.

The BvgS/BvgA phosphorelay system of pathogenic Bordetellae: structure, function and evolution.

In the genus Bordetella several important human and animal pathogens are classified with B. pertussis, the etiological agent of whooping cough, being medically the most relevant. In these bacteria expression of the most important virulence factors including several toxins, adhesins and colonization factors is controlled by a single master regulatory two-component system, the BvgS/BvgA system. This system represents a paradigm of a complex phosphorelay system that mediates a fine-tuned transcriptional response resulting in different expression levels of virulence factors during different stages of the infection process. In this chapter the current knowledge about signal perception and the molecular basis of differential gene expression controlled by a single two-component system is discussed.

Nutritional upgrading for omnivorous carpenter ants by the endosymbiont Blochmannia.

BACKGROUND: Carpenter ants (genus Camponotus) are considered to be omnivores. Nonetheless, the genome sequence of Blochmannia floridanus, the obligate intracellular endosymbiont of Camponotus floridanus, suggests a function in nutritional upgrading of host resources by the bacterium. Thus, the strongly reduced genome of the endosymbiont retains genes for all subunits of a functional urease, as well as those for biosynthetic pathways for all but one (arginine) of the amino acids essential to the host. RESULTS: Nutritional upgrading by Blochmannia was tested in 90-day feeding experiments with brood-raising in worker-groups on chemically defined diets with and without essential amino acids and treated or not with antibiotics. Control groups were fed with cockroaches, honey water and Bhatkar agar. Worker-groups were provided with brood collected from the queenright mother-colonies (45 eggs and 45 first instar larvae each). Brood production did not differ significantly between groups of symbiotic workers on diets with and without essential amino acids. However, aposymbiotic worker groups raised significantly less brood on a diet lacking essential amino acids. Reduced brood production by aposymbiotic workers was compensated when those groups were provided with essential amino acids in their diet. Decrease of endosymbionts due to treatment with antibiotic was monitored by qRT-PCR and FISH after the 90-day experimental period. Urease function was confirmed by feeding experiments using 15N-labelled urea. GC-MS analysis of 15N-enrichment of free amino acids in workers revealed significant labelling of the non-essential amino acids alanine, glycine, aspartic acid, and glutamic acid, as well as of the essential amino acids methionine and phenylalanine. CONCLUSION: Our results show that endosymbiotic Blochmannia nutritionally upgrade the diet of C. floridanus hosts to provide essential amino acids, and that it may also play a role in nitrogen recycling via its functional urease. Blochmannia may confer a significant fitness advantage via nutritional upgrading by enhancing competitive ability of Camponotus with other ant species lacking such an endosymbiont. Domestication of the endosymbiont may have facilitated the evolutionary success of the genus Camponotus.

Identification and regulation of expression of a gene encoding a filamentous hemagglutinin-related protein in Bordetella holmesii.

BACKGROUND: Bordetella holmesii is a human pathogen closely related to B. pertussis, the etiological agent of whooping cough. It is able to cause disease in immunocompromised patients, but also whooping cough-like symptoms in otherwise healthy individuals. However, virtually nothing was known so far about the underlying virulence mechanisms and previous attempts to identify virulence factors related to those of B. pertussis were not successful. RESULTS: By use of a PCR approach we were able to identify a B. holmesii gene encoding a protein with significant sequence similarities to the filamentous hemagglutinin (FHA) of B. avium and to a lesser extent to the FHA proteins of B. pertussis, B. parapertussis, and B. bronchiseptica. For these human and animal pathogens FHA is a crucial virulence factor required for successful colonization of the host. Interestingly, the B. holmesii protein shows a relatively high overall sequence similarity with the B. avium protein, while sequence conservation with the FHA proteins of the human and mammalian pathogens is quite limited and is most prominent in signal sequences required for their export to the cell surface. In the other Bordetellae expression of the fhaB gene encoding FHA was shown to be regulated by the master regulator of virulence, the BvgAS two-component system. Recently, we identified orthologs of BvgAS in B. holmesii, and here we show that this system also contributes to regulation of fhaB expression in B. holmesii. Accordingly, the purified BvgA response regulator of B. holmesii was shown to bind specifically in the upstream region of the fhaB promoter in vitro in a manner similar to that previously described for the BvgA protein of B. pertussis. Moreover, by deletion analysis of the fhaB promoter region we show that the BvgA binding sites are relevant for in vivo transcription from this promoter in B. holmesii. CONCLUSION: The data reported here show that B. holmesii is endowed with a factor highly related to filamentous hemagglutinin (FHA), a prominent virulence factor of the well characterized pathogenic Bordetellae. We show that like in the other Bordetellae the virulence regulatory BvgAS system is also involved in the regulation of fhaB expression in B. holmesii. Taken together these data indicate that in contrast to previous notions B. holmesii may in fact make use of virulence mechanisms related to those described for the other Bordetellae.