Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement.

Among horizontally acquired symbioses, the mechanisms underlying microbial strain- and species-level specificity remain poorly understood. Here, confocal-microscopy analyses and genetic manipulation of the squid-vibrio association revealed quantitative differences in a symbiont's capacity to interact with the host during initial engagement. Specifically, dominant strains of Vibrio fischeri, 'D-type', previously named for their dominant, single-strain colonization of the squid's bioluminescent organ, were compared with 'S-type', or 'sharing', strains, which can co-colonize the organ. These D-type strains typically: (i) formed aggregations of 100s-1000s of cells on the light-organ surface, up to 3 orders of magnitude larger than those of S-type strains; (ii) showed dominance in co-aggregation experiments, independent of inoculum size or strain proportion; (iii) perturbed larger areas of the organ's ciliated surface; and, (iv) appeared at the pore of the organ approximately 4×s more quickly than S-type strains. At least in part, genes responsible for biofilm synthesis control the hyperaggregation phenotype of a D-type strain. Other marine vibrios produced relatively small aggregations, while an array of marine Gram-positive and -negative species outside of the Vibrionaceae did not attach to the organ's surface. These studies provide insight into the impact of strain variation on early events leading to establishment of an environmentally acquired symbiosis.

Bactericidal Permeability-Increasing Proteins Shape Host-Microbe Interactions.

We characterized bactericidal permeability-increasing proteins (BPIs) of the squid Euprymna scolopes, EsBPI2 and EsBPI4. They have molecular characteristics typical of other animal BPIs, are closely related to one another, and nest phylogenetically among invertebrate BPIs. Purified EsBPIs had antimicrobial activity against the squid's symbiont, Vibrio fischeri, which colonizes light organ crypt epithelia. Activity of both proteins was abrogated by heat treatment and coincubation with specific antibodies. Pretreatment under acidic conditions similar to those during symbiosis initiation rendered V. fischeri more resistant to the antimicrobial activity of the proteins. Immunocytochemistry localized EsBPIs to the symbiotic organ and other epithelial surfaces interacting with ambient seawater. The proteins differed in intracellular distribution. Further, whereas EsBPI4 was restricted to epithelia, EsBPI2 also occurred in blood and in a transient juvenile organ that mediates hatching. The data provide evidence that these BPIs play different defensive roles early in the life of E. scolopes, modulating interactions with the symbiont.IMPORTANCE This study describes new functions for bactericidal permeability-increasing proteins (BPIs), members of the lipopolysaccharide-binding protein (LBP)/BPI protein family. The data provide evidence that these proteins play a dual role in the modulation of symbiotic bacteria. In the squid-vibrio model, these proteins both control the symbiont populations in the light organ tissues where symbiont cells occur in dense monoculture and, concomitantly, inhibit the symbiont from colonizing other epithelial surfaces of the animal.

Partner choice and fidelity stabilize coevolution in a Cretaceous-age defensive symbiosis.

Many insects rely on symbiotic microbes for survival, growth, or reproduction. Over evolutionary timescales, the association with intracellular symbionts is stabilized by partner fidelity through strictly vertical symbiont transmission, resulting in congruent host and symbiont phylogenies. However, little is known about how symbioses with extracellular symbionts, representing the majority of insect-associated microorganisms, evolve and remain stable despite opportunities for horizontal exchange and de novo acquisition of symbionts from the environment. Here we demonstrate that host control over symbiont transmission (partner choice) reinforces partner fidelity between solitary wasps and antibiotic-producing bacteria and thereby stabilizes this Cretaceous-age defensive mutualism. Phylogenetic analyses show that three genera of beewolf wasps (Philanthus, Trachypus, and Philanthinus) cultivate a distinct clade of Streptomyces bacteria for protection against pathogenic fungi. The symbionts were acquired from a soil-dwelling ancestor at least 68 million years ago, and vertical transmission via the brood cell and the cocoon surface resulted in host-symbiont codiversification. However, the external mode of transmission also provides opportunities for horizontal transfer, and beewolf species have indeed exchanged symbiont strains, possibly through predation or nest reuse. Experimental infection with nonnative bacteria reveals that--despite successful colonization of the antennal gland reservoirs--transmission to the cocoon is selectively blocked. Thus, partner choice can play an important role even in predominantly vertically transmitted symbioses by stabilizing the cooperative association over evolutionary timescales.

Maternal and environmental effects on symbiont-mediated antimicrobial defense.

Bacteria produce a remarkable diversity of bioactive molecules with antimicrobial properties. Despite the importance of such compounds for human medicine, little is known about the factors influencing antibiotic production in natural environments. Recently, several insects have been found to benefit from symbiont-produced antimicrobial compounds for defense against pathogenic microbes. In the European beewolf, Philanthus triangulum (Hymenoptera, Crabronidae), bacteria of the genus Streptomyces provide protection against pathogens by producing antimicrobials on the larval cocoon during hibernation, thereby significantly enhancing the survival probability of the beewolf larva. To investigate the effects of abiotic and biotic factors on antibiotic production, we exposed beewolf cocoons to different environmental conditions and quantified the amount of Streptomyces-produced antibiotics by using gas chromatography/mass spectrometry (GC/MS). The results revealed no significant influence of temperature, humidity, or pathogen load on the antibiotic amount, indicating that antibiotic production is not affected by current environmental conditions but rather may be optimized to serve as a reliable long-term protection during the unpredictable phase of beewolf hibernation. However, the amount of antibiotics was positively correlated with the symbiont population size on the cocoon, which in turn is affected by the number of Streptomyces cells provided by the mother into the brood cell. Additionally, we found a positive correlation between the amount of hydrocarbons and the number and length of bacterial cells in the antennal gland secretion, suggesting that maternal investment affects symbiont growth and, thus, antibiotic production on the larval cocoon.

Dynamics of symbiont-mediated antibiotic production reveal efficient long-term protection for beewolf offspring.

BACKGROUND: Insects have evolved a wide range of mechanisms to defend themselves and their offspring against antagonists. One of these strategies involves the utilization of antimicrobial compounds provided by symbiotic bacteria to protect the host or its nutritional resources from pathogens and parasites. In the symbiosis of the solitary digger wasp, Philanthus triangulum (Hymenoptera, Crabronidae), the bacterial symbiont 'Candidatus Streptomyces philanthi' defends the developing larvae against pathogens by producing a mixture of at least nine antimicrobial substances on the cocoon surface. This antibiotic cocktail inhibits the growth of a broad range of detrimental fungi and bacteria, thereby significantly enhancing the offspring's survival probability. RESULTS: Here we show that the production of antimicrobial compounds by the beewolf symbionts is confined to the first two weeks after cocoon spinning, leading to a high concentration of piericidins and streptochlorin on the cocoon surface. Expression profiling of housekeeping, sporulation, and antibiotic biosynthesis genes indicates that antibiotic production coincides with morphological differentiation that enables the symbionts to survive the nutrient-limited conditions on the beewolf cocoon. The antibiotic substances remain stable on the cocoon surface for the entire duration of the beewolf's hibernation period, demonstrating that the compounds are resistant against environmental influences. CONCLUSIONS: The antibiotic production by the beewolf symbionts serves as a reliable protection for the wasp offspring against pathogenic microorganisms during the long and unpredictable developmental phase in the subterranean brood cells. Thus, the beewolf-Streptomyces symbiosis provides one of the rare examples of antibiotics serving as an efficient defense in the natural environment and may aid in devising new strategies for the utilization of antibiotic combination therapies in human medicine against increasingly resistant bacterial and fungal pathogens.

Botulinum toxin: A noninvasive option for the symptomatic treatment of salivary gland stenosis - a case report.

BACKGROUND: A man diagnosed with Stensen's duct stenosis exhibited recurrent parotid swelling, invariably during meals. Previous parotid duct dilations and percutaneous radiotherapy were ineffective. Botulinum toxin (BTX) injections were injected into the affected gland to regulate salivary flow and reduce parotid swelling. METHODS: BTX (22.5 units) was injected into the affected gland. A second treatment with 30 units BTX was carried out 7 weeks later. Two further injections followed after 4 months, respectively. The results were scored by the patient and evaluated in an examination. RESULTS: The patient reported the disappearance of parotid swelling after 2 weeks of injections. This effect was maintained for 5 weeks after the first treatment and for 4 months after the following 2 treatments. There were no side effects. CONCLUSION: Here we introduce BTX as a therapeutic option for the treatment of salivary duct stenosis when other therapies are ineffective and before opting for gland extirpation.