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Mosquito-borne diseases are a major global health threat. Traditional morphological or molecular methods for identifying mosquito species often require specialized expertise or expensive laboratory equipment. The use of convolutional neural networks (CNNs) to identify mosquito species based on images may offer a promising alternative, but their practical implementation often remains limited. This study explores the applicability of CNNs in classifying mosquito species. It compares the efficacy of body and wing depictions across three image collection methods: a smartphone, macro-lens attached to a smartphone and a professional stereomicroscope. The study included 796 specimens of four morphologically similar Aedes species, Aedes aegypti, Ae. albopictus, Ae. koreicus and Ae. japonicus japonicus. The findings of this study indicate that CNN models demonstrate superior performance in wing-based classification 87.6% (95% CI: 84.2-91.0) compared to body-based classification 78.9% (95% CI: 77.7-80.0). Nevertheless, there are notable limitations of CNNs as they perform reliably across multiple devices only when trained specifically on those devices, resulting in an average decline of mean accuracy by 14%, even with extensive image augmentation. Additionally, we also estimate the required training data volume for effective classification, noting a reduced requirement for wing-based classification compared to body-based methods. Our study underscores the viability of both body and wing classification methods for mosquito species identification while emphasizing the need to address practical constraints in developing accessible classification systems., (© 2024. The Author(s).)

Background: Mosquito host feeding patterns are an important factor of the species-specific vector capacity determining pathogen transmission routes. Culex pipiens s.s./Cx. torrentium are competent vectors of several arboviruses, such as West Nile virus and Usutu virus. However, studies on host feeding patterns rarely differentiate the morphologically indistinguishable females., Methods: We analyzed the host feeding attraction of Cx. pipiens and Cx. torrentium in host-choice studies for bird, mouse, and a human lure. In addition, we summarized published and unpublished data on host feeding patterns of field-collected specimens from Germany, Iran, and Moldova from 2012 to 2022, genetically identified as Cx. pipiens biotype pipiens, Cx. pipiens biotype molestus, Cx. pipiens hybrid biotype pipiens × molestus, and Cx. torrentium, and finally put the data in context with similar data found in a systematic literature search., Results: In the host-choice experiments, we did not find a significant attraction to bird, mouse, and human lure for Cx. pipiens pipiens and Cx. torrentium. Hosts of 992 field-collected specimens were identified for Germany, Iran, and Moldova, with the majority determined as Cx. pipiens pipiens, increasing the data available from studies known from the literature by two-thirds. All four Culex pipiens s.s./Cx. torrentium taxa had fed with significant proportions on birds, humans, and nonhuman mammals. Merged with the data from the literature from 23 different studies showing a high prevalence of blood meals from birds, more than 50% of the blood meals of Cx. pipiens s.s. were identified as birds, while up to 39% were human and nonhuman mammalian hosts. Culex torrentium fed half on birds and half on mammals. However, there were considerable geographical differences in the host feeding patterns., Conclusions: In the light of these results, the clear characterization of the Cx. pipiens s.s./Cx. torrentium taxa as ornithophilic/-phagic or mammalophilic/-phagic needs to be reconsidered. Given their broad host ranges, all four Culex taxa could potentially serve as enzootic and bridge vectors., (© 2024. The Author(s).)

Background: Mosquitoes are important vectors of pathogens. They are usually collected with CO 2 -baited traps and subsequently identified by morphology. This procedure is very time-consuming. Automatic counting traps could facilitate timely evaluation of the local risk for mosquito-borne pathogen transmission or decision-making on vector control measures, but the counting accuracy of such devices has rarely been validated in the field., Methods: The Biogents (BG)-Counter 2 automatically counts mosquitoes by discriminating the size of captured objects directly in the field and transmits the data to a cloud server. To assess the accuracy of this counting device, 27 traps were placed at 19 sampling sites across Germany and used in daily, weekly or bimonthly intervals from April until October 2021. The BG-Counter 2 was attached to a CO 2 -trap (BG-Pro trap = CO 2 -Pro) and the same trap was converted to also attract gravid mosquitoes (upside-down BG-Pro trap with a water container beneath = CO 2 -Pro-gravid). All captured mosquitoes were identified by morphology. The number of females (unfed and gravid), mosquito diversity and the number of identified specimens in relation to the counting data of the BG-Counter were compared between the two trapping devices to evaluate sampling success and counting accuracy., Results: In total 26,714 mosquitoes were collected during 854 trap days. The CO 2 -Pro-gravid trap captured significantly more mosquitoes per trap day for all specimens, gravid females and non-gravid females, while there was no difference in the mosquito diversity. The linear model with the captured mosquitoes as a response and the counted specimens as a predictor explained only a small degree of the variation within the data (R 2  = 0.16), but per individual trap the value could reach up to 0.62 (mean R 2  = 0.23). The counting accuracy for the daily samples had a significant positive correlation with sample size, resulting in higher accuracy for the CO 2 -Pro-gravid trap and higher accuracy for sites and sampling months with high mosquito abundance., Conclusions: While the accuracy of the BG-Counter 2 is quite low, the device is able to depict mosquito phenology and provide information about local population dynamics., (© 2024. The Author(s).)

Two Wolbachia strains, w Mel and w AlbB, have been transinfected into Aedes aegypti mosquitoes for population replacement with the aim of reducing dengue transmission. Epidemiological data from various endemic sites suggest a pronounced decrease in dengue transmission after implementing this strategy. In this study, we investigated the impact of the Wolbachia strains w Mel and w AlbB on Ae. aegypti fitness in a common genetic background. We found that Ae. aegypti females infected with the w Mel strain exhibited several significant differences compared with those infected with the w AlbB strain. Specifically, w Mel-infected females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the w AlbB strain. Conversely, the w AlbB strain showed only mild negative effects when compared with Wolbachia -uninfected specimens. These differential effects on Ae. aegypti fitness following infection with either w Mel or w AlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations in endemic settings. Further research is needed to better understand the underlying mechanisms responsible for these differences in fitness effects and their potential impact on the long-term efficacy of Wolbachia -based dengue control programs.IMPORTANCEThe transmission of arboviruses such as dengue, Zika, and chikungunya is on the rise globally. Among the most promising strategies to reduce arbovirus burden is the release of one out of two strains of Wolbachia -infected Aedes aegypti : w Mel and w AlbB. One critical aspect of whether this approach will succeed involves the fitness cost of either Wolbachia strains on mosquito life history traits. For instance, we found that w Mel-infected Ae. aegypti females laid significantly fewer eggs, ingested a lower amount of blood, had a reduced egg production rate, and exhibited a decreased Wolbachia density at a later age compared with mosquitoes infected with the w AlbB strain. Conversely, the w AlbB strain showed only mild negative effects when compared with Wolbachia -uninfected specimens. These differential effects on mosquito fitness following infection with either w Mel or w AlbB may have important implications for the success of population replacement strategies in invading native Ae. aegypti populations., Competing Interests: The authors declare no conflict of interest.

Accurate species identification is crucial to assess the medical relevance of a mosquito specimen, but requires intensive experience of the observers and well-equipped laboratories. In this proof-of-concept study, we developed a convolutional neural network (CNN) to identify seven Aedes species by wing images, only. While previous studies used images of the whole mosquito body, the nearly two-dimensional wings may facilitate standardized image capture and reduce the complexity of the CNN implementation. Mosquitoes were sampled from different sites in Germany. Their wings were mounted and photographed with a professional stereomicroscope. The data set consisted of 1155 wing images from seven Aedes species as well as 554 wings from different non-Aedes mosquitoes. A CNN was trained to differentiate between Aedes and non-Aedes mosquitoes and to classify the seven Aedes species based on grayscale and RGB images. Image processing, data augmentation, training, validation and testing were conducted in python using deep-learning framework PyTorch. Our best-performing CNN configuration achieved a macro F1 score of 99% to discriminate Aedes from non-Aedes mosquito species. The mean macro F1 score to predict the Aedes species was 90% for grayscale images and 91% for RGB images. In conclusion, wing images are sufficient to identify mosquito species by CNNs., (© 2024. The Author(s).)

Background: Aedes japonicus japonicus (Theobald, 1901) and Aedes koreicus (Edwards, 1917) have rapidly spread in Europe over the last decades. Both species are very closely related and occur in sympatry. Females and males are difficult to distinguish. However, the accurate species discrimination is important as both species may differ in their vectorial capacity and spreading behaviour. In this study, we assessed the potential of geometric wing morphometrics as alternative to distinguish the two species., Methods: A total of 147 Ae. j. japonicus specimens (77 females and 70 males) and 124 Ae. koreicus specimens (67 females and 57 males) were collected in southwest Germany. The left wing of each specimen was removed, mounted and photographed. The coordinates of 18 landmarks on the vein crosses were digitalised by a single observer. The resulting two-dimensional dataset was used to analyse the differences in the wing size (i.e. centroid size) and wing shape between Ae. j. japonicus and Ae. koreicus using geometric morphometrics. To analyse the reproducibility of the analysis, the landmark collection was repeated for 20 specimens per sex and species by two additional observers., Results: The wing size in female Ae. koreicus was significantly greater than in Ae. j. japonicus but did not differ significantly for males. However, the strong overlap in wing size also for the females would not allow to discriminate the two species. In contrast, the wing shape clustering was species specific and a leave-one-out validation resulted in a reclassification accuracy of 96.5% for the females and 91.3% for the males. The data collected by different observers resulted in a similar accuracy, indicating a low observer bias for the landmark collection., Conclusions: Geometric wing morphometrics provide a reliable and robust tool to distinguish female and male specimens of Ae. j. japonicus and Ae. koreicus., (© 2023. The Author(s).)

The spatial distribution of mosquito species in the course of globalization and climate warming is highly dynamic. Different studies have demonstrated the spread and establishment of thermophilic mosquito species, potentially increasing the prevalence of 'nuisance' mosquitoes and the local transmission of pathogens. Here we report the first recorded sampling of Anopheles hyrcanus in Wrocław, southwest Poland. This is the most northern detection of this species to date in Europe. Future spread and population development of this potential vector of malaria parasites, viruses or zoonotic helminths, such as Dirofilaria spp., must be monitored carefully. Potential factors underlying the spread of this species are discussed., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: The impact of the arbovirus vector Aedes aegypti is of major concern for global public health as the viruses that it transmits affect millions of people each year worldwide. Originating in Africa, Ae. aegypti has now spread throughout much of the world. While the genetic makeup of Ae. aegypti in the New World has been extensively studied, there is limited knowledge on its genetic diversity in Africa, particularly at a microgeographical level., Methods: We investigated mitochondrial cytochrome oxidase I of four Ae. aegypti populations from Benin and employed wing morphometric analyses as a cost-effective and reliable tool to explore population structure. Our sampling encompassed various areas of Benin, from the southern to the northern borders of the country, and included urban, semi-urban, and sylvatic sites., Results: We observed a notable level of genetic diversity (haplotype diversity of 0.8333) and nucleotide diversity (0.00421986), and identified seven distinct haplotypes. Sylvatic and semi-urban sites exhibited a greater number of haplotypes compared to urban sites. Utilizing 18 wing landmarks, we calculated the centroid size, which revealed significant variation among the three landscape types. However, principal component analysis, employed to assess wing shape variation, did not demonstrate significant differences between populations based on landscape type., Conclusions: Our findings indicate substantial genetic and morphological diversity among Ae. aegypti populations in Benin, and provide insight into important biological characteristics of these populations with respect to their potential to transmit viruses. To the best of our knowledge, this is the first study undertaken in Africa to integrate genetics with morphology to analyse the population structure of the major arbovirus vector Ae. aegypti., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Culex pipiens s.s./ Culex torrentium belong to the most widespread mosquito taxa in Europe and are the main vectors of Sindbis, West Nile and Usutu virus. The adult overwintering females can act as reservoir for these arthropod-borne viruses (arboviruses), thus contributing to their local persistence when transmission cycles are interrupted during the winter. However, the main overwintering sites of Cx. torrentium are unknown. In a study from 2017, 3455 Cx. pipiens s.s./ Cx. torrentium specimens were collected from abandoned animal burrows in Poznan, Poland. These specimens were retrospectively identified to species-level with a PCR assay, which revealed Cx. torrentium as dominant species (> 60%). Motivated by these results, we conducted a field study from February to July 2022 to systematically analyse the overwintering site patterns of Cx. pipiens s.s./ Cx. torrentium . Mosquitoes were sampled using pipe traps in abandoned animal burrows ( n  = 20) and with aspirators in nearby anthropogenic overwintering sites ( n  = 23). All Cx. pipiens s.s./ Cx. torrentium were screened for Flaviviridae RNA. In total, 4710 mosquitoes of five different taxa were collected from anthropogenic sites. 3977 of them were identified as Cx. p. pipiens / Cx. torrentium ( Cx. p. pipiens : 85%, Cx torrentium : 1%, pools with both species: 14%). In contrast, only Cx. p. pipiens / Cx. torrentium (1688 specimens) were collected from animal burrows dominated by Cx. torrentium (52%), followed by pools with both species (40%) and Cx. p pipiens (8%). A single pool of 10 Cx. torrentium specimens collected from an animal burrow was positive for Usutu virus. The detection of Usutu virus demonstrates that Cx. torrentium can act as winter reservoir for arboviruses. Abandoned animal burrows may by the primary overwintering site for the species and should be considered in future surveillance programmes, when sampling overwintering mosquitoes., Competing Interests: The authors declare that they have no competing interests., (© 2023 The Authors.)

Knowledge of the hibernation site preferences and the factors which influence winter survival in these hibernation sites may enhance understanding of mosquito population dynamics after winter and how arboviruses persist in temperate regions. Our study quantified the number of adult overwintering mosquitoes in cellars and aboveground constructions and analyzed survival rates in relation to the environmental conditions in these sites. During the winters 2016/2017 and 2018/2019, 149 different constructions in Northwest Germany were sampled for mosquitoes. Mosquitoes were detected in 44% of the cellars and in 33% of the aboveground constructions. Culex p. pipiens Linnaeus was the most abundant species in cellars, whereas high numbers of Anopheles messeae Falleroni were collected from a single barn. Subsequently, an enclosure study was conducted during 2019/2020. Overwintering field-collected Cx. p. pipiens and An. messeae were divided into groups with or without fructose availability, and placed in cages with different man-made hibernations sites, where temperature and relative humidity were recorded hourly. For both species, increasing mean temperatures (5-16°C) but not mean relative humidity (58-94%) were correlated with winter mortality rates of the mosquitoes. The lipid measurements were greater and mortality rates were lower when both species were provided fructose. Larger specimens (determined by wing length) stored more lipids, and in Cx. p pipiens, but not in An. messeae, survival probability of large specimens was significantly greater than for small females. Mosquitoes showed a distinct pattern in the selection of overwintering sites, while temperature was an important driver for survival., (© The Author(s) 2022. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Environmental temperature is a key driver for the transmission risk of mosquito-borne pathogens. Epidemiological models usually relate to temperature data from standardized weather stations, but these data may not capture the relevant scale where mosquitoes experience environmental temperatures. As mosquitoes are assumed to spend most of their lifetime in resting sites, we analysed mosquito resting site patterns and the associated temperatures in dependence on the resting site type, resting site height and the surrounding land use., Methods: The study was conducted in 20 areas in near-natural habitats in Germany. Ten areas were studied in 2017, and another 10 in 2018. Each study area consisted of three sampling sites, where we collected mosquitoes and microclimatic data in artificial (= garden pop-up bags) and natural resting sites at three height levels between 0 and 6 m. Land use of the study sites was characterized as forest and meadows based on reclassified information of the CORINE (Coordination of Information on the Environment) Land Cover categories. The hourly resting site temperatures and the data from the nearest weather station of the German meteorological service were used to model the duration of the extrinsic incubation period (EIP) of mosquito-borne pathogens., Results: Anopheles, Culex and Culiseta preferred artificial resting sites, while Aedes were predominantly collect in natural resting sites. Around 90% of the mosquitoes were collected from resting sites below 2 m. The mosquito species composition did not differ significantly between forest and meadow sites. Mean resting site temperatures near the ground were approximately 0.8 °C lower than at a height of 4-6 m, which changed the predicted mean EIP up to 5 days at meadow and 2 days at forest sites. Compared with temperature data from standardized weather stations, the resting site temperatures near the ground would prolong the mean estimated EIP 4 days at forest sites and 2 days at meadow sites., Conclusions: The microclimate of mosquito resting sites differs from standardized meteorological data, which can influence the transmission of mosquito-borne pathogens. In a near-natural environment, colder temperatures at mosquitoes' preferred resting sites near the ground would prolong the EIP of mosquito-borne pathogens relative to data from weather stations., (© 2022. The Author(s).)

Mosquitoes (Diptera: Culicidae) use certain resting sites during their inactive phase. The microclimatic conditions of these resting sites might affect their physiology and vectorial capacity. In this study, we combined a field and a laboratory study to investigate the natural resting site and temperature preferences of mosquitoes. The field study was conducted at a forest close to Oldenburg (Lower Saxony, Germany) from May to October 2018. Mosquitoes were collected in five different natural habitats with seven replicates each. Temperature was recorded hourly at each site. Significantly more mosquitoes were collected in deadwood (predominantly Culiseta morsitans/fumipennis) and shaded herb layer (predominantly Aedes species) compared to unshaded herb layer or broadleaf and coniferous trees. GLMMs revealed resting site habitats as the best predictor to explain the observed preference patterns, but microclimatic conditions are also involved in mosquito resting site selection. Most mosquitoes were collected at resting sites with relatively colder and more stable temperatures. In concert, laboratory choice experiments with a thermal gradient ring demonstrated that Cs. morsitans/fumipennis avoid temperatures over 30 °C. Understanding the small-scaled resting site preferences and the related microclimatic conditions can improve mosquito collection techniques and refine the prediction of mosquito-borne pathogen transmission., (© 2021 The Authors. Medical and Veterinary Entomology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.)

Accurate species identification is the prerequisite to assess the relevance of mosquito specimens, but is often hindered by missing or damaged morphological features. The present study analyses the applicability of wing geometric morphometrics as a low-cost and practical alternative to identify native mosquitoes in Germany. Wing pictures were collected for 502 female mosquitoes of five genera and 19 species from 80 sampling sites. The reliable species identification based on interspecific wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross validation revealed an overall accuracy of 99% for the genus and 90% for the species identification. Misidentifications were mainly due to three pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Similar to the results of the morphometric analysis, the same problematic three Aedes-pairs clustered, but most other species could be well separated. Overall, our study underpins that morphometric wing analysis is a robust tool for reliable mosquito identification, which reach the accuracy of COI barcoding.

Salinization of freshwater ecosystems as a result of human activities represents a global threat for ecosystems' integrity. Whether different sources of salinity with their differing ionic compositions lead to variable effects in ecosystem functioning is unknown. Therefore, the present study assessed the impact of dryland- (50μS/cm to 11,000μS/cm) and coalmine-induced (100μS/cm to 2400μS/cm) salinization on the leaf litter breakdown, with focus on microorganisms as main decomposer, in two catchments in New South Wales, Australia. The breakdown of Eucalyptus camaldulensis leaves decreased with increasing salinity by up to a factor of three. Coalmine salinity, which is characterised by a higher share of bicarbonates, had a slightly but consistently higher breakdown rate at a given salinity relative to dryland salinity, which is characterised by ionic proportions similar to sea water. Complementary laboratory experiments supported the stimulatory impact of sodium bicarbonates on leaf breakdown when compared to sodium chloride or artificial sea salt. Furthermore, microbial inoculum from a high salinity site (11,000μS/cm) yielded lower leaf breakdown at lower salinity relative to inoculum from a low salinity site (50μS/cm). Conversely, inoculum from the high salinity site was less sensitive towards increasing salinity levels relative to inoculum from the low salinity site. The effects of the different inoculum were the same regardless of salt source (sodium bicarbonate, sodium chloride and artificial sea salt). Finally, the microorganism-mediated leaf litter breakdown was most efficient at intermediate salinity levels (≈500μS/cm). The present study thus points to severe implications of increasing salinity intensities on the ecosystem function of leaf litter breakdown, while the underlying processes need further scrutiny., (Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.)

Bacterial pathogens utilize the chaperone-usher pathway to assemble extracellular multi-subunit fibers essential for virulence. The periplasmic chaperone facilitates the initial folding of fiber subunits but then traps them in activated folding transition states. Chaperone dissociation releases the folding energy that drives subunit incorporation into the fiber, which grows through a pore formed by the outer-membrane usher.

The assembly of type 1 pili on the surface of uropathogenic Escherichia coli proceeds via the chaperone-usher pathway. Chaperone-subunit complexes interact with one another via a process termed donor strand complementation whereby the G1beta strand of the chaperone completes the immunoglobulin (Ig) fold of the pilus subunit. Chaperone-subunit complexes are targeted to the usher, which forms a channel across the outer membrane through which pilus subunits are translocated and assembled into pili via a mechanism known as donor strand exchange. This is a mechanism whereby chaperone uncapping from a subunit is coupled with the simultaneous assembly of the subunit into the pilus fiber. Thus, in the pilus fiber, the N-terminal extension of every subunit completes the Ig fold of its neighboring subunit by occupying the same site previously occupied by the chaperone. Here, we investigated details of the donor strand exchange assembly mechanism. We discovered that the information necessary for targeting the FimC-FimH complex to the usher resides mainly in the FimH protein. This interaction is an initiating event in pilus biogenesis. We discovered that the ability of an incoming subunit (in a chaperone-subunit complex) to participate in donor strand exchange with the growing pilus depended on a previously unrecognized function of the chaperone. Furthermore, the donor strand exchange assembly mechanism between subunits was found to be necessary for subunit translocation across the outer membrane usher.

Periplasmic chaperones direct the assembly of adhesive, multi-subunit pilus fibers that play critical roles in bacterial pathogenesis. Pilus assembly occurs via a donor strand exchange mechanism in which the N-terminal extension of one subunit replaces the chaperone G(1) strand that transiently occupies a groove in the neighboring subunit. Here, we show that the chaperone primes the subunit for assembly by holding the groove in an open, activated conformation. During donor strand exchange, the subunit undergoes a topological transition that triggers the closure of the groove and seals the N-terminal extension in place. It is this topological transition, made possible only by the priming action of the chaperone that drives subunit assembly into the fiber.

Bacterial pili assembled by the chaperone-usher pathway can mediate microbial attachment, an early step in the establishment of an infection, by binding specifically to sugars present in host tissues. Recent work has begun to reveal the structural basis both of chaperone function in the biogenesis of these pili and of bacterial attachment.

A fundamental question in molecular biology is how proteins fold into domains that can serve as assembly modules for building up large macromolecular structures. The biogenesis of pili on the surface of Gram-negative bacteria requires the orchestration of a complex process that includes protein synthesis, folding via small chaperones, secretion, and assembly. The results presented here support the hypothesis that pilus subunit folding and biogenesis proceed via mechanisms termed donor strand complementation and donor strand exchange. Here we show that the steric information necessary for pilus subunit folding is not contained in one polypeptide sequence. Rather, the missing information is transiently donated by a strand of a small chaperone to allow folding. Providing the missing information for folding, via a 13-amino acid peptide extension to the C-terminal end of a pilus subunit, resulted in the production of a protein that no longer required the chaperone to fold. This mechanism of small periplasmic chaperone function described here deviates from classical hsp60 chaperone-assisted folding.

Haemophilus influenzae haemagglutinating pili are surface appendages that promote attachment to host cells and facilitate respiratory tract colonization, an essential step in the pathogenesis of disease. In contrast to other well-characterized forms of pili, H. influenzae haemagglutinating pili are two-stranded helical structures. Nevertheless, haemagglutinating pili are assembled by a pathway that involves a periplasmic chaperone and an outer membrane usher, analogous to the prototype pathway involved in the biogenesis of Escherichia coli P pili. In this study, we performed site-directed mutagenesis of the H. influenzae HifB chaperone and HifA major pilus subunit at positions homologous to sites important for chaperone-subunit interactions and subunit oligomerization in P pili. Mutations at putative subunit binding pocket residues in HifB or at the penultimate tyrosine in HifA abolished formation of HifB-HifA periplasmic complexes, whereas mutations at the -14 glycine in HifA had no effect on HifB-HifA interactions but abrogated HifA oligomerization. To define further the constraints of the interaction between HifA and HifB, we examined the interchangeability of pilus gene cluster components from H. influenzae type b strain Eagan (hifA-hifEEag) and the related H. influenzae biogroup aegyptius strain F3031 (hifA-hifEF3031). Functional pili were assembled both with HifAEag and the strain F3031 gene cluster and with HifAF3031 and the strain Eagan gene cluster, underscoring the flexibility of the H. influenzae chaperone/usher pathway in incorporating HifA subunits with significant sequence diversity. To gain additional insight into the interactive surfaces of HifA and HifB, we aligned HifA sequences from 20 different strains and then modelled the HifA structure based on the recently crystallized PapD-PapK complex. Analysis of the resulting structure revealed high levels of sequence conservation in regions predicted to interact with HifB, and maximal sequence diversity in regions potentially exposed on the surface of assembled pili. These results suggest broad applicability of structure-function relationships identified in studies of P pili, including the concepts of donor strand complementation and donor strand exchange. In addition, they provide insight into the structure of HifA and suggest a basis for antigenic variation in H. influenzae haemagglutinating pili.

The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis. PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus. Subunits lack the seventh *gb-strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand. Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding. In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor. Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site., (Copyright 2000 Academic Press.)

Gram-negative bacteria produce a diverse array of pili that mediate microbe-microbe and host-pathogen interactions important in the development of disease. The structural and functional characterization of these organelles, particularly their role in triggering signals in both the bacterium and the host upon attachment, has begun to reveal the molecular mechanisms of bacterial diseases.

Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.

A panel of murine monoclonal antibodies was generated against the extracellular domain of the human platelet-derived growth factor (PDGF) beta receptor (PDGFRbeta). These antibodies were assayed for both the ability to inhibit binding of PDGF BB to PDGFRbeta+ cells as well as the capacity to inhibit PDGF BB-mediated mitogenesis. As expected, all antibodies that could prevent PDGF BB binding also inhibited mitogenesis. However one antibody (M4TS.11), with no detectable ability to inhibit PDGF BB binding, was a potent inhibitor of proliferation induced by PDGF BB. Further characterization indicated that M4TS.11 impaired PDGFRbeta dimerization, revealing the mechanism by which it prevented PDGF BB-mediated mitogenesis. Using domain deletion mutants of the extracellular portion of PDGFRbeta, the determinant recognized by this antibody was localized to the fourth extracellular domain of PDGFRbeta, indicating that this domain, which is not involved in ligand binding, actively participates in receptor dimerization and signal transduction. The M4TS.11 antibody could also inhibit PDGF BB-mediated proliferation of responsive cells from both the baboon and the rabbit, indicating the determinant recognized by the antibody is not limited to humans and making it possible to use this antibody to evaluate the therapeutic benefit of interfering with PDGF in animal models of human disease.