Upstream CtrA-binding sites both induce and repress pilin gene expression in Caulobacter crescentus.

Pili are bacterial surface structures important for surface adhesion. In the alphaproteobacterium Caulobacter crescentus, the global regulator CtrA activates transcription of roughly 100 genes, including pilA which codes for the pilin monomer that makes up the pilus filament. While most CtrA-activated promoters have a single CtrA-binding site at the - 35 position and are induced at the early to mid-predivisional cell stage, the pilA promoter has 3 additional upstream CtrA-binding sites and it is induced at the late predivisional cell stage. Reporter constructs where these additional sites were disrupted by deletion or mutation led to increased activity compared to the WT promoter. In synchronized cultures, these mutations caused pilA transcription to occur approximately 20 min earlier than WT. The results suggested that the site overlapping the - 35 position drives pilA gene expression while the other upstream CtrA-binding sites serve to reduce and delay expression. EMSA experiments showed that the - 35 Site has lower affinity for CtrA∼P compared to the other sites, suggesting binding site affinity may be involved in the delay mechanism. Mutating the upstream inhibitory CtrA-binding sites in the pilA promoter caused significantly higher numbers of pre-divisional cells to express pili, and phage survival assays showed this strain to be significantly more sensitive to pilitropic phage. These results suggest that pilA regulation evolved in C. crescentus to provide an ecological advantage within the context of phage infection.

The PulE ATPase is required for twitching motility and DNA donation during Thermus thermophilus transjugation.

Thermus thermophilus can acquire DNA through natural competence and through transjugation, a mechanism that involves a two-step process of DNA secretion (push) and DNA internalization (pull) between mating cells of related species. The natural competence apparatus (NCA) is required in the recipient mate for the pull step. However, how the DNA exits of the donor cell is only partially known. The putative DNA translocase TdtA, encoded in mobile genetic element ICETh1 of T. thermophilus HB27, was shown to be required for DNA donation as reported by (Blesa et al. 2017a). This ring-shaped hexameric ATPase binds to the membrane and likely interacts with yet unknown secretory components that allow the extrusion of DNA through the membrane; thus, a genetic screening to identify additional putative secretory components was performed. Here, we describe that mutants in gene TT_C1844, which encodes a putative AAA-ATPase named PulE, do not synthesize the recently described "narrow" type 4 pili required for twitching motility and made up of the major PilA5 pilin. Concomitantly, pulE mutants also exhibited DNA donation defects during transjugation, suggesting a role of narrow pili in the donation process. However, single pilA5 null mutants still function as DNA donors in transjugation experiments, so we conclude that the need for PulE in transjugation is independent of its role in narrow pili synthesis and twitching motility.

Pili Subunit PilA Contributes to the Cytoadhesion of Glaesserella Parasuis to Host Cells and Provides Immunoprotection.

Glaesserella parasuis (G. parasuis) is commonly located in the upper respiratory tract of pigs as an opportunistic pathogen. It can cause Glässer's disease, which leads to serious economic losses in the swine industry. The occurrence of the disease is often linked with the adhesion and colonization of the pathogen. The PilA pilus subunit is important for adhesion to the host, twitching motility, and biofilm formation in many bacteria. However, no research has focused on the function of PilA in G. parasuis. To further reveal the pathogenesis of G. parasuis and to search for subunit vaccine candidates, we investigated whether PilA could adhere to cells and provide immune protection. A bioinformatic analysis showed that the protein secondary structure of the G. parasuis PilA was similar to that of Haemophilus influenzae (HI). Cell adhesion, ELISA, and far-Western blotting showed that rPilA could bind porcine-derived, porcine kidney-15 (PK-15) cells, swine tracheal epithelial cells (STECs), and the extracellular matrix components fibronectin (FN) and laminin (LN). An immunogenicity analysis showed that recombinant PilA (rPilA) reacted specifically with convalescent and hyperimmune serum. Importantly, purified rPilA elicited a strong immune response and conferred robust protection against challenges with serovar 5 G. parasuis in mice. These results suggested that the PilA protein might help G. parasuis adhere to host cells by binding to FN and LN, and its immunogenicity establishes it as a promising, novel subunit vaccine candidate against infections with G. parasuis. IMPORTANCE G. parasuis is one of the most prevalent bacterial infections in swine production and can lead to huge economic losses around the world. A full understanding of colonization and immunity with G. parasuis infections will be essential in disease control. In this study, the PilA protein, which is a common virulence factor in other bacteria that mediates adherence to the host, was assessed. The results suggested that the PilA protein of G. parasuis can mediate adhesion to host cells through FN and LN, which provides a new idea for the study of the pathogenicity of G. parasuis. Furthermore, fimbriae usually have high immunogenicity. Immunogenicity and protective capacity results showed that the use of this recombinant PilA antigen might be a promising candidate vaccine antigen with which to prevent G. parasuis infections.

Papillary Intralymphatic Angioendothelioma in a Child With PIK3CA-Related Overgrowth Spectrum: Implication of PI3K Pathway in the Vascular Tumorigenesis.

Papillary intralymphatic angioendothelioma (PILA) is an extremely rare vascular tumor and its pathogenesis is unknown. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth spectrum (PROS) is a heterogeneous group of disorders caused by mosaicism for activating mutations of PIK3CA and characterized by asymmetric overgrowth, skeletal anomalies, skin lesions, and vascular malformations. An association between PILA and PROS has not been known. We report a case of PILA involving the spleen of a young girl with the clinical and molecular diagnosis of PROS. Sequencing of the patient's germ-line DNA detected a pathogenic PIK3CA variant c.1357G>A in 10.6% of alleles. Splenectomy revealed a 4-cm tumor composed of ectatic lymphatics with intraluminal papillary projections, consistent with PILA. The tumor cells showed immunohistochemical expression of CD31, CD34, ERG, FLI-1, PROX1, and caldesmon, while D2-40 was negative. The latter may suggest that the tumor derived from an endothelial precursor arrested in the final steps of lymphothelial differentiation, in keeping with the known role of the PIK3CA-governed molecular pathway in the progression of vascular progenitors to mature endothelial cells. The data implicates PIK3CA in the pathogenesis of PILA and broadens the spectrum of phenotypic expressions of PROS.

Identification of major and cross-reactive allergens of local freshwater snail (Pila polita) and the impact of thermal and non-thermal food processing on allergen stability.

BACKGROUND: Snail allergy is rare but can be fatal. Pila polita, a freshwater snail, was considered as a popular exotic food, particularly in tropical countries, and consumed in processed forms. Thus, the purpose of this study was to identify the major and cross-reactive allergens of P. polita and to determine the impact of food processing on the allergen stability. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionated raw snail extract to approximately 24 protein bands, between 9 and 245 kDa. The prominent band at 33 kDa was detected in all raw and processed snail extracts. Immunoblotting tests of the raw extract demonstrated 19 immunoglobulin E (IgE)-binding proteins, and four of them, at 30, 35, 42 and 49 kDa, were revealed as the major IgE-binding proteins of P. polita. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified the 49 and 42 kDa major allergens as actin, whereas the 30 and 35 kDa major allergens were identified as tropomyosin. Immunoblotting revealed that the raw snail had more allergenic proteins than the processed snail. The degree of allergenicity in decreasing order was raw > brine pickled> boiled > roasted > fried > vinegar pickled. The presence of cross-reactivity between P. polita and the shellfish tested was exhibited with either no, complete, or partial inhibitions. CONCLUSION: Actin and tropomyosin were identified as the major and cross-reactive allergens of P. polita among local patients with snail allergy. Those major allergens are highly stable to high temperatures, acidic pH, and high salt, which might played a crucial role in snail allergy in Malaysia. © 2023 Society of Chemical Industry.

Spatio-temporal changes in oxidative stress physiology parameters in apple snail Pila globosa as a function of soil Mg, Ca, organic carbon and aquatic physico-chemical factors.

Information on the oxidative stress physiology parameters (OSPP) in general and as a function of the fluctuation of Mg, Ca and organic carbon present in soil and aquatic physico-chemical factors such as pH, temperature and salinity in particular are scanty in the amphibious snail Pila globosa. A spatio-temporal analysis of redox metabolism (as OSPP) followed by discriminant function analysis of the obtained data were performed in P. globosa sampled from the east-coasts of Odisha state, India (mostly along the Bay of Bengal) for environmental health assessment purposes. Results revealed that the OSPP are susceptible to seasonal synergistic variation of soil and physico-chemical factors. Overall, lipid peroxidation, total antioxidant capacity, activities of catalase, glutathione reductase had positive correlation whereas ascorbic acid, the reduced glutathione and the activity of superoxide dismutase had non-significant correlation with the soil Mg, Ca, organic carbon, and pH, temperature and salinity of water. In the summer season, the snails had a marked 51.83% and 26.41% higher lipid peroxidation level and total antioxidative activity as compared to the other seasons. Spatial variation of OSPP indicates that snails residing away from the Bay of Bengal coast had at least 4.4% lower antioxidant level in winter and 30% higher lipid peroxide levels in summer as compared to the rest of the sampling sites. Results on OSPP in P. globosa may be useful for monitoring the ecotoxic effects of environment using molluscs in general and P. globosa in particular.

Nontypeable Haemophilus influenzae Type IV Pilus Mediates Augmented Adherence to Rhinovirus-Infected Human Airway Epithelial Cells.

Human rhinovirus (hRV) is frequently detected in the upper respiratory tract, and symptomatic infection is associated with an increased nasopharyngeal bacterial load, with subsequent development of secondary bacterial diseases. Nontypeable Haemophilus influenzae (NTHI) is a commensal bacterial species of the human nasopharynx; however, in the context of prior or concurrent upper respiratory tract viral infection, this bacterium commonly causes multiple diseases throughout the upper and lower respiratory tracts. The present study was conducted to determine the mechanism(s) by which hRV infection promotes the development of NTHI-induced diseases. We showed that hRV infection of polarized primary human airway epithelial cells resulted in increased adherence of NTHI, due in part to augmented expression of CEACAM1 and ICAM1, host cell receptors to which NTHI binds via engagement of multiple adhesins. Antibody blockade of these host cell receptors significantly reduced NTHI adherence. With a specific focus on the NTHI type IV pilus (T4P), which we have previously shown binds to ICAM1, an essential adhesin and virulence determinant, we next showed that T4P-directed antibody blockade significantly reduced NTHI adherence to hRV-infected airway cells and, further, that expression of this adhesin was required for the enhanced adherence observed. Collectively, these data provide a mechanism by which "the common cold" promotes diseases due to NTHI, and they add further support for the use of PilA (the majority subunit of T4P) as a vaccine antigen, since antibodies directed against PilA are expected to limit the notably increased bacterial load associated with hRV coinfection and thereby to prevent secondary NTHI-induced diseases of the respiratory tract.

A trivalent vaccine consisting of "flagellin A+B and pilin" protects against Pseudomonas aeruginosa infection in a murine burn model.

Pseudomonas aeruginosa is a common nosocomial pathogen in burn patients, and rapidly achieves antibiotic resistance, and thus, developing an effective vaccine is critically important for combating P. aeruginosa infection. Flagella and pili play important roles in colonization of P. aeruginosa at the burn wound site and its subsequent dissemination to deeper tissue and organs. In the present study, we evaluated protective efficacy of a trivalent vaccine containing flagellins A and B (FlaA + FlaB) + pilin (PilA) in a murine burn model of infection. "FlaA + FlaB + PilA" induced greater protection in P. aeruginosa murine burn model than the single components alone, and it showed broad immune protection against P. aeruginosa strains. Immunization with "FlaA + FlaB + PilA" induced strong opsonophagocytic antibodies and resulted in reduced bacterial loads, systemic IL-12/IL-10 cytokine expression, and increased survival after challenge with three times lethal dose fifty (LD50) of P. eruginosa strains. Moreover, the protective efficacy of "FlaA + FlaB + PilA" vaccination was largely attributed to specific antibodies. Taken together, these data further confirm that the protective effects of "FlaA + FlaB + PilA" vaccine significantly enhance efficacy compared with antibodies against either mono or divalent antigen, and that the former broadens the coverage against P. eruginosa strains that express two of the three antigens.

Expression of the Nontypeable Haemophilus influenzae Type IV Pilus Is Stimulated by Coculture with Host Respiratory Tract Epithelial Cells.

The type IV pilus (Tfp) of nontypeable Haemophilus influenzae (NTHI) mediates adherence, colonization, motility, and biofilm formation, and the major protein subunit, PilA, is a promising vaccine candidate. Thus, it is crucial to understand how Tfp expression is regulated within the microenvironments of the human nasopharynx, which NTHI colonizes asymptomatically, and the more distal regions of the respiratory tract where NTHI-induced diseases occur. Here, we examined the effects of coculture of NTHI with human airway epithelial cells and heme availability on Tfp expression at temperatures typical of the human nasopharynx (34°C) or warmer anatomical sites during infection (37°C). Tfp expression was estimated by pilA promoter activity, pilA gene expression, and relative abundances of PilA and pilin protein. The results revealed that at both temperatures, NTHI cocultured with airway epithelial cells demonstrated significantly greater expression of pilA, PilA/pilin protein, and likely, fully assembled Tfp than NTHI cultured on an abiotic surface. Because NTHI is a heme auxotroph, we hypothesized that availability of heme from host cells might be a signal for Tfp expression. Thereby, we cultured NTHI in iron-limited medium, and we observed that supplementation with heme significantly increased pilA promoter activity. Collectively, our data suggested that NTHI Tfp expression was stimulated by soluble factor(s) released by epithelial cells, which are present in all microenvironments of the respiratory tract. The expression of this target antigen under conditions that mimic the human airway strongly supports the rationale for the use of PilA as a vaccine immunogen to prevent NTHI-induced diseases of the respiratory tract.

A Protein E-PilA Fusion Protein Shows Vaccine Potential against Nontypeable Haemophilus influenzae in Mice and Chinchillas.

PE-PilA is a fusion protein composed of immunologically relevant parts of protein E (PE) and the majority subunit of the type IV pilus (PilA), two major antigens of nontypeable Haemophilus influenzae (NTHi). Here we report on the preclinical evaluation of PE-PilA as a vaccine antigen. The immunogenic potential of the PE and PilA within the fusion was compared with that of isolated PE and PilA antigens. When injected intramuscularly into mice, the immunogenicity of PE within the fusion was equivalent to that of isolated PE, except when it was formulated with alum. In contrast, in our murine models PilA was consistently found to be more immunogenic as a subentity of the PE-PilA fusion protein than when it was injected as an isolated antigen. Following immunization with PE-PilA, anti-PE antibodies demonstrated the same capacity to inhibit the binding of PE to vitronectin as those induced after PE immunization. Likewise, PE-PilA-induced anti-PilA antibodies inhibited the formation of NTHi biofilms and disrupted established biofilms in vitro These experiments support the immunogenic equivalence between fused PE-PilA and isolated PE and PilA. Further, the potential of PE-PilA immunization against NTHi-induced disease was evaluated. After intranasal NTHi challenge, colonization of the murine nasopharynx significantly dropped in animals formerly immunized with PE-PilA, and in chinchillas, signs of otitis media were significantly reduced in animals that had received anti-PE-PilA antibodies. Taken together, our data support the use of PE-PilA as an NTHi vaccine antigen.

Design and Characterization of Protein E-PilA, a Candidate Fusion Antigen for Nontypeable Haemophilus influenzae Vaccine.

Nontypeable Haemophilus influenzae (NTHi) is a pathogen known for being a frequent cause of acute otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In the present study, a vaccine antigen based on the fusion of two known NTHi adhesive proteins, protein E (PE) and a pilin subunit (PilA), was developed. The quality of the combined antigen was investigated through functional, biophysical, and structural analyses. It was shown that the PE and PilA individual structures are not modified in the PE-PilA fusion and that PE-PilA assembles as a dimer in solution, reflecting PE dimerization. PE-PilA was found to bind vitronectin by enzyme-linked immunosorbent assay, as isolated PE does. Disulfide bridges were conserved and homogeneous, which was determined by peptide mapping and top-down analysis of PE, PilA, and PE-PilA molecules. Finally, the PE-PilA crystal showed a PE entity with a three-dimensional (3D) structure similar to that of the recently published isolated PE, while the structure of the PilA entity was similar to that of a 3D model elaborated from two other type 4 pilin subunits. Taken together, our observations suggest that the two tethered proteins behave independently within the chimeric molecule and display structures similar to those of the respective isolated antigens, which are important characteristics for eliciting optimal antibody-mediated immunity. PE and PilA can thus be further developed as a single fusion protein in a vaccine perspective, in the knowledge that tethering the two antigens does not perceptibly compromise the structural attributes offered by the individual antigens.

Artyfechinostomum sufrartyfex Trematode Infections in Children, Bihar, India.

Eating raw or insufficiently cooked mollusks is a known risk factor for human echinostomiasis. We confirmed identification of Artyfechinostomum sufrartyfex trematodes as the causative agent of disease among 170 children in northern Bihar, India. We also identified the snail Pila globosa as a potential source of infections in the study area.

Biological synthesis of high-conductive pili in aerobic bacterium Pseudomonas aeruginosa.

Bioelectrical nanowires as ecomaterials have great potential on environmental applications. A wide range of bacteria can express type IV pili (T4P), which are long protein fibers assembled from PilA. The T4P of Geobacter sulfurreducens are well known as "microbial nanowires," yet T4P of Pseudomonas aeruginosa (PaT4P) was believed to be poorly conductive. P. aeruginosa is an aerobic and electrochemically active bacterium. Its T4P have been known to be responsible for surface attachment, twitching motility and biofilm formation. Here, we show that PaT4P can be highly conductive while assembled by a truncated P. aeruginosa PilA (PaPilA) containing only N-terminus 61 amino acids. Furthermore, increasing the number of aromatic amino acids in the PaPilA1-61 significantly enhances the conductivity of pili and the bioelectricity output of P. aeruginosa in microbial fuel cell system, suggesting a potential application of PaT4P as a conductive nanomaterial. The N-terminal region of PilA from diverse eubacteria is highly conserved, implying a general way to synthesize highly conductive microbial nanowires and to increase the bioelectricity output of microbial fuel cell.

AmpuBase: a transcriptome database for eight species of apple snails (Gastropoda: Ampullariidae).

BACKGROUND: Gastropoda, with approximately 80,000 living species, is the largest class of Mollusca. Among gastropods, apple snails (family Ampullariidae) are globally distributed in tropical and subtropical freshwater ecosystems and many species are ecologically and economically important. Ampullariids exhibit various morphological and physiological adaptations to their respective habitats, which make them ideal candidates for studying adaptation, population divergence, speciation, and larger-scale patterns of diversity, including the biogeography of native and invasive populations. The limited availability of genomic data, however, hinders in-depth ecological and evolutionary studies of these non-model organisms. RESULTS: Using Illumina Hiseq platforms, we sequenced 1220 million reads for seven species of apple snails. Together with the previously published RNA-Seq data of two apple snails, we conducted de novo transcriptome assembly of eight species that belong to five genera of Ampullariidae, two of which represent Old World lineages and the other three New World lineages. There were 20,730 to 35,828 unigenes with predicted open reading frames for the eight species, with N50 (shortest sequence length at 50% of the unigenes) ranging from 1320 to 1803 bp. 69.7% to 80.2% of these unigenes were functionally annotated by searching against NCBI's non-redundant, Gene Ontology database and the Kyoto Encyclopaedia of Genes and Genomes. With these data we developed AmpuBase, a relational database that features online BLAST functionality for DNA/protein sequences, keyword searching for unigenes/functional terms, and download functions for sequences and whole transcriptomes. CONCLUSIONS: In summary, we have generated comprehensive transcriptome data for multiple ampullariid genera and species, and created a publicly accessible database with a user-friendly interface to facilitate future basic and applied studies on ampullariids, and comparative molecular studies with other invertebrates.

A Short Protocol for Gene Knockout and Complementation in Xylella fastidiosa Shows that One of the Type IV Pilin Paralogs (PD1926) Is Needed for Twitching while Another (PD1924) Affects Pilus Number and Location.

Twitching motility is one of the major virulence factors of the plant-pathogenic bacterium Xylella fastidiosa, and it is mediated by type IV pili (TFP) that are present at one of the cell poles. Genome analysis of X. fastidiosa showed the presence of at least four paralogs of the gene pilA, which encodes the TFP major pilin subunit. However, whether all of these paralogs have a functional role in TFP structure and function is unknown. Here, using a short and reliable protocol based on overlap extension PCR and natural transformation, deletion mutants of two pilA paralogs (pilA1 PD1924 and pilA2 PD1926) were generated in two X. fastidiosa subsp. fastidiosa strains, WM1-1 and TemeculaL, followed by assessment of twitching motility and biofilm formation. Deletion of pilA2 caused loss of twitching motility, whereas deletion of pilA1 did not influence twitching motility but caused hyperpiliation and extended distribution of TFP along the sides of the cell. Loss of twitching motility due to pilA2 deletion was restored when a wild-type copy of the pilA2 gene was added at a neutral site in the genome of mutants in both wild-type backgrounds. This study demonstrates that PCR templates generated by overlap extension PCR can be successfully used to rapidly generate gene knockouts and perform genetic complementation in X. fastidiosa, and that twitching motility in X. fastidiosa is controlled by regulating the transcription of the major pilin subunit, pilA2IMPORTANCE The bacterial plant pathogen Xylella fastidiosa causes incurable diseases in multiple hosts, including grape, citrus, and blueberry. Historically restricted to the Americas, it was recently found to cause epidemics in olives in Italy and to infect other hosts in Europe and Asia. In this study, we report a short protocol to create deletion and complemented mutants using fusion PCR and natural transformation. We also determined the distinct function of two pilin paralogs, the main structural component of TFP involved in twitching motility, which allows this bacterium to move inside the xylem vessels against the flow. One of the paralogs is needed for twitching movement, whereas the other does not have an effect on motility but influences the number and position of TFP. Since twitching motility is fundamental for the virulence of this xylem-limited bacterium, this study contributes to the understanding of the regulation of virulence by this pathogen.

Comparative genomics of host adaptive traits in Xanthomonas translucens pv. graminis.

BACKGROUND: Xanthomonas translucens pathovars differ in their individual host ranges among Poaceae. As the causal agent of bacterial wilt in Italian ryegrass (Lolium multiflorum Lam.), X. translucens pv. graminis (Xtg) is one of the most important bacterial pathogens in temperate grassland regions. The genomes of six Xtg strains from Switzerland, Norway, and New Zealand were sequenced in order to gain insight into conserved genomic traits from organisms covering a wide geographical range. Subsequent comparative analysis with previously published genome data of seven non-graminis X. translucens strains including the pathovars arrhenatheri, poae, phlei, cerealis, undulosa, and translucens was conducted to identify candidate genes linked to the host adaptation of Xtg to Italian ryegrass. RESULTS: Phylogenetic analysis revealed a tight clustering of Xtg strains, which were found to share a large core genome. Conserved genomic traits included a non-canonical type III secretion system (T3SS) and a type IV pilus (T4P), which both revealed distinct primary structures of the pilins when compared to the non-graminis X. translucens strains. Xtg-specific traits that had no homologues in the other X. translucens strains were further found to comprise several hypothetical proteins, a TonB-dependent receptor, transporters, and effector proteins as well as toxin-antitoxin systems and DNA methyltransferases. While a nearly complete flagellar gene cluster was identified in one of the sequenced Xtg strains, phenotypic analysis pointed to swimming-deficiency as a common trait of the pathovar graminis. CONCLUSION: Our study suggests that host adaptation of X. translucens pv. graminis may be conferred by a combination of pathovar-specific effector proteins, regulatory mechanisms, and adapted nutrient acquisition. Sequence deviations of pathogen-associated molecular patterns (PAMPs), as observed for the pilins of the T4P and T3SS, are moreover likely to impede perception by the plant defense machinery and thus facilitate successful host colonization of Italian ryegrass.

Antibodies raised against divalent type b flagellin and pilin provide effective immunotherapy against Pseudomonas aeruginosa infection of mice with burn wounds.

Burn wound infections caused by multidrug-resistant Pseudomonas aeruginosa strains are a serious challenge to therapy because of the complex pathogenesis and paucity of new effective antibiotics. Therefore, there is renewed interest in developing antibody-based therapeutic strategies. Immunotherapy strategies typically target selected virulence factors that are expressed by the majority of clinical strains of P. aeruginosa, particularly because virulence factors mediate infection. Here we used a murine model of burn wound infection to evaluate the efficacy of antibodies raised against the divalent type b flagellin and PilA (flagellin b + PilA), as acute virulence factors, to prevent and treat infection. Antibodies to flagellin b + PilA exhibited superior synergistic effects that improved opsono-phagocytosis and cell invasion compared with antibodies to each monovalent flagellin b or PilA. Further, when used for prophylaxis, the antibodies against flagellin b + PilA and combined therapeutic and prophylactic regimens markedly improved the survival of mice infected with disparate P. aeruginosa strains from 91.6% to 100% compared with treatment using imipenem. Therefore, antibodies against flagellin b + PilA interfere with the activities of their respective cognate individual target antigens and enhance coverage against clinical strains of P. aeruginosa that may not express one of these two virulence factors.

Artyfechinostomum malayanum: Metacercariae Encysted in Pila sp. Snails Purchased from Phnom Penh, Cambodia.

The metacercariae of Artyfechinostomum malayanum (Leiper, 1911) Mendheim, 1943 were discovered in Pila sp. snails purchased from a market in Phnom Penh, Cambodia. They were isolated from the snails using the artificial digestion technique and were orally fed to 2 hamsters, 1 rat, and 2 mice to obtain the adult flukes. The metacercariae were round, 145-165 µm in diameter, having a cyst wall of 6-10 µm in thickness, a head collar and collar spines, and characteristic features of excretory granules. Adult flukes were recovered in the small intestines of the animals at days 14 and 32 post infection and were morphologically observed using a light microscope and a scanning electron microscope. They were plump or elongated, ventrally curved, 6.0-8.1×1.6-2.0 mm in size, and characterized by the head collar bearing 43 collar spines, including 5 end group ones on each side, a long cirrus sac extending beyond the posterior margin of the ventral sucker, a submedian ovary, and 2 deeply lobed testes. Eggs in uteri were operculate, ovoid to ellipsoid, and 120-135×68-75 µm in size. In scanning electron microscopy, the head collar was prominent with collar spines looking like horns. Scale-like tegumental spines were densely distributed on the ventral surface between the head collar and ventral sucker. Sensory papillae were distributed mainly on the tegument around suckers. By this study, it has been first confirmed that the life cycle of A. malayanum exists in Cambodia.

Passive immunization against Pseudomonas aeruginosa recombinant PilA in a murine burn wound model.

Pseudomonas aeruginosa type IV pili have an essential role in twitching motility, colonization and biofilm formation. In this study, we investigated the efficacy of intraperitoneal administration of rabbit anti-recombinant PilA (anti-r-PilA) immunoglobulin G (IgG) against P. aeruginosa infection in a mouse burn-wound model. After burn and infection, mortality rate was assessed in all mice, and that of mice passively immunized with rabbit anti-r-PilA IgG was compared to non-immunized mice. Bacterial quantities in the skin and internal organs were measured to determine the level of systemic infection. Results showed that passive immunotherapy with anti-r-PilA IgG protected the burned mice infected with P. aeruginosa strains, PAO1 and the clinical isolate (CI). Anti-r-PilA antibodies enhanced the opsonophagocytosis of these strains. Moreover, the administration of anti-r-PilA IgG was also successful in reducing the bacterial burden in infected mice. The reduction of systemic bacterial spread increased the survival rate of passively immunized mice. Findings of this study revealed an improved survival rate of 62.5%, thus confirming the protective effect of anti-r-PilA IgG.

Protective effect of pilin protein with alum+naloxone adjuvant against acute pulmonary Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa is an important opportunistic human pathogen that causes a wide variety of severe nosocomial infections. Type IV pili of P. aeruginosa are made up of polymerized pilin that aids in bacterial adhesion, biofilm formation and twitching motility. The aim of this study was to evaluate the efficacy of alum and naloxone (alum+NLX) as an adjuvant for P. aeruginosa recombinant PilA (r-PilA) as a vaccine candidate in the improvement of humoral and cellular immunity. Primary immunization with r-PilA in combination with alum+NLX followed by two booster shots was sufficient to generate robust cellular and humoral responses, which were Th1 and Th2 type responses consisting of IgG1 and IgG2a subtypes. Analysis of the cytokine response among immunized mice showed an increased production of IL-4, INF-γ and IL-17 by splenocytes upon stimulation by r-PilA. These sera were also able to reduce bacterial load in the lung tissue of challenged mice. The reduction of systemic bacterial spread resulted in increased survival rates in challenged immunized mice. In conclusion, immunization with r-PilA combined with alum+NLX evokes cellular and humoral immune responses, which play an important role in providing protection against acute P. aeruginosa lung infection among immunized mice.