Understanding regulation of the host-mediated gut symbiont population and the symbiont-mediated host immunity in the Riptortus-Burkholderia symbiosis system

Valuable insect models have tremendously contributed to our understanding of innate immunity and symbiosis. Bean bug, Riptortus pedestris, is a useful insect symbiosis model due to harboring cultivable monospecific gut symbiont, genus Burkholderia. Bean bug is a hemimetabolous insect whose immunity is not well-understood. However, we recently identified three major antimicrobial peptides of Riptortus and examined the relationship between gut symbiosis and host immunity. We found that the presence of Burkholderia gut symbiont positively affects Riptortus immunity. From studying host regulation mechanisms of symbiont population, we revealed that the symbiotic Burkholderia cells are much more susceptible to Riptortus immune responses than the cultured cells. We further elucidated that the immune-susceptibility of the Burkholderia gut symbionts is due to the drastic change of bacterial cell envelope. Finally, we show that the immune-susceptible Burkholderia symbionts are able to prosper in host owing to the suppression of immune responses of the symbiotic midgut.     

Oral Immunization with Recombinant Lactobacillus acidophilus Expressing the Adhesin Hp0410 of Helicobacter pylori Induces Mucosal and Systemic Immune Responses

Helicobacter pylori infection is relatively common worldwide and is closely related to gastric mucosa-associated lymphoid tissue (MALT) lymphoma, chronic gastritis, and stomach ulcers. Therefore, a safe and effective method for preventing H. pylori infection is urgently needed. Given that developing an effective vaccine against H. pylori is one of the best alternatives, H. pylori adhesin Hp0410 was expressed in the food-grade bacterium Lactobacillus acidophilus. The recombinant live bacterial vaccine was then used to orally vaccinate mice, and the immunoprotective effects of Hp0410-producing strains were investigated. H. pylori colonization in the stomach of mice immunized with the recombinant L. acidophilus was significantly reduced, in comparison with that in control groups. Furthermore, mucosal secretory IgA antibodies were elicited in the mucosal tissue of mice immunized with the recombinant bacteria, and specific anti-Hp0410 IgG responses were also detected in mouse serum. There was a significant increase in the level of protection against gastric Helicobacter infection following a challenge with H. pylori Sydney strain 1 (SS1). Our results collectively indicate that adhesin Hp0410 is a promising candidate vaccine antigen, and recombinant L. acidophilus expressing Hp0410 is likely to constitute an effective, low-cost, live bacterial vaccine against H. pylori.     

Bacteria of the genus Dyella can chronically colonise the airways of patients with cystic fibrosis and elicit a pronounced antibody response

A patient with cystic fibrosis became chronically colonised with an unusual non-fermenting Gram-negative rod that could be cultured on Burkholderia cepacia selective agar. Phenotypic characterisation by VITEK-2 suggested identification as Elizabethkingia meningoseptica, however 16S rRNA gene sequencing revealed it belonged to a putative novel species of genus Dyella. Thirty months after the initial detection, the patient produced a high level of precipitating antibodies against the bacterium.     

Colonization of Cecum Is Important for Development of Persistent Infection by Yersinia pseudotuberculosis

Yersiniosis is a human disease caused by the bacterium Yersinia pseudotuberculosis or Yersinia enterocolitica. The infection is usually resolved but can lead to postinfectious sequelae, including reactive arthritis and erythema nodosum. The commonly used Yersinia mouse infection model mimics acute infection in humans to some extent but leads to systemic infection and eventual death. Here, we analyzed sublethal infection doses of Y. pseudotuberculosis in mice in real time using bioluminescent imaging and found that infections using these lower doses result in extended periods of asymptomatic infections in a fraction of mice. In a search for the site for bacterial persistence, we found that the cecum was the primary colonization site and was the site where the organism resided during a 115-day infection period. Persistent infection was accompanied by sustained fecal shedding of cultivable bacteria. Cecal patches were identified as the primary site for cecal colonization during persistence. Y. pseudotuberculosis bacteria were present in inflammatory lesions, in localized foci, or as single cells and also in neutrophil exudates in the cecal lumen. The chronically colonized cecum may serve as a reservoir for dissemination of infection to extraintestinal sites, and a chronic inflammatory state may trigger the onset of postinfectious sequelae. This novel mouse model for bacterial persistence in cecum has potential as an investigative tool to unveil a deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection.     

Elimination of symbiotic Aeromonas spp. from the intestinal tract of the medicinal leech,Hirudo medicinalis,using ciprofloxacin feeding

The use of the medicinal leech (Hirudo medicinalis) in promoting venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially in plastic and reconstructive surgery, has been complicated by infections caused by Aeromonas spp. These are leech endosymbionts for which patients undergoing hirudotherapy frequently receive systemic chemoprophylaxis. In order to evaluate the possibility of rendering leeches safe for use on patients, H. medicinalis were fed artificially with a 2 g/L arginine solution (used as a phagostimulant) supplemented with ciprofloxacin (100 mg/L). Aeromonads were detected in 57 out of 80 control leeches (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001). Treated leeches survived for up to 4 months. Tested weekly, 61% of these leeches took human blood for at least 4 weeks after treatment and all remained negative for aeromonads. All water samples in which leeches were kept before treatment were contaminated with Aeromonas spp.; none were detected in any of the NaCl/arginine solutions with which treated animals were fed. Molecular characterization of two phenotypically distinct isolates using gyrB sequencing showed that one clustered tightly with A. veronii and the other was closely related to A. media. Other environmental bacteria and fungi were isolated from 26.5% of treated leeches that had taken a blood meal 1–4 weeks after treatment. Ciprofloxacin reduced the number of leech-associated aeromonads to undetectable levels for extended periods. Most treated leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy.     

Symbiosis of Aeromonas veronii Biovar sobria and Hirudo medicinalis, the Medicinal Leech: a Novel Model for Digestive Tract Associations

Hirudo medicinalis, the medicinal leech, is applied postoperatively in modern medicine. Infections by Aeromonas occur in up to 20% of patients unless a preemptive antibiotic treatment is administered. The associated infections demonstrate the need for a better understanding of the digestive tract flora of H. medicinalis. Early studies reported the presence of a single bacterial species in the digestive tract and suggested that these bacteria were endosymbionts contributing to the digestion of blood. In this study, we cultivated bacteria from the digestive tract and characterized them biochemically. The biochemical test results identified the isolates as Aeromonas veronii biovar sobria. This species identification was supported by sequence comparison of a variable region of the genes coding for 16S rRNA. In a colonization assay, a rifampin-resistant derivative of a symbiotic isolate was fed in a blood meal to H. medicinalis. The strain colonized the digestive tract rapidly and reached a concentration similar to that of the native bacterial flora. For the first 12 h, the in vivo doubling time was 1.2 h at 23°C. After 12 h, at a density of 5 × 10⁷ CFU/ml, the increase in viable counts ceased, suggesting a dramatic reduction in the bacterial growth rate. Two human fecal isolates, identified as Aeromonas hydrophila and A. veronii biovar sobria, were also able to colonize the digestive tract. These data demonstrate that the main culturable bacterium in the crop of H. medicinalis is A. veronii biovar sobria and that the medicinal leech can be used as a model for digestive tract association of Aeromonas species.     

Molting-associated suppression of symbiont population and up-regulation of antimicrobial activity in the midgut symbiotic organ of the Riptortus–Burkholderia symbiosis

The majority of insects possess symbiotic bacteria. Since symbiont titers can affect host phenotypes of biological importance, host insects are expected to evolve some mechanisms for regulating symbiont population. Here we report that, in the Riptortus–Burkholderia gut symbiosis, titers of the beneficial symbiont transiently decrease at the pre-molt stages in host development. This molting-associated suppression of the symbiont population is coincident with the increase of antimicrobial activity in the symbiotic midgut, which is observed in both symbiotic and aposymbiotic insects. Two genes, pyrrhocoricin-like antimicrobial peptide and c-type lysozyme, exhibit significantly increased expression in the symbiotic midgut at the pre-molt stages. These results suggest that the molting-associated up-regulation of antimicrobial activity in the symbiotic midgut represents a physiological mechanism of the host insect to regulate symbiosis, which is presumably for defending molting insects against injury and infection and/or for allocating symbiont-derived energy and resources to host molting.     

Phylogenetic Placement of Rickettsiae from the Ticks Amblyomma americanum and Ixodes scapularis

A rickettsial isolate (isolate MOAa) belonging to the spotted fever group (SFG) was obtained from the lone star tick Amblyomma americanum. We used PCR to characterize the genes for the rickettsial outer membrane proteins rOmpA and rOmpB. We sequenced the PCR products (domains I of both the rompA gene and the rompB gene) of MOAa and WB-8-2, another rickettsial isolate from A. americanum. To place MOAa and WB-8-2 and two other nonpathogenic isolates (Rickettsia rickettsii Hlp2 and Rickettsia montana M5/6) with respect to their putative sister species, we included them in a phylogenetic analysis of 9 Rickettsia species and 10 Rickettsia strains. Our phylogenetic results implied three evolutionary lineages of SFG rickettsiae and that WB-8-2 and MOAa were most closely related to R. montana. New World isolates were not the most closely related to each other (they did not form a clade). Rather, our results supported four independent origins (introductions) of rickettsiae into North America from different Old World regions. The results of our phylogenetic analysis did not support the hypothesis of a stable coevolution of rickettsiae and their tick hosts. Finally, we examined the rompA gene of a nonpathogenic rickettsial symbiont isolated from the tick Ixodes scapularis. In a phylogenetic analysis, the symbiont was placed as the sister to R. montana and its isolates. The relationship of this symbiont to R. montana raised questions as to the potential origin of pathogenic SFG rickettsiae from nonpathogenic tick symbionts, or vice versa.     

MAPT H1 haplotype is associated with enhanced α-synuclein deposition in dementia with Lewy bodies

The microtubule-associated protein tau (MAPT) H1 haplotype has been identified as a genetic risk factor for synucleinopathies. However, whether it modulates tau or α-synuclein pathology remains unknown. Our aim was to investigate the relationship between MAPT haplotypes and pathologic aggregates of tau and α-synuclein in pathologically confirmed cases of dementia with Lewy bodies (DLB). Twenty-two cases fulfilling clinical and neuropathological criteria for DLB were included. Clinical and neuropathological data were collected, and APOE and MAPT genotypes were determined. Tau and α-synuclein pathology was assessed semiquantitatively in 17 brain areas and total scores were calculated. DLB H1/H1 (n = 12) and H2 carriers (n = 10) did not differ in demographics, clinical variables, concomitant Alzheimer's pathology, or APOE genotype. Total α-synuclein scores were significantly increased in the H1/H1 group (p = 0.011), largely due to an increase in brainstem regions. This difference was driven by an increase in Lewy bodies and diffuse and punctuate cytoplasmatic α-synuclein aggregates (p = 0.007 and p = 0.025 respectively). These findings provide a mechanistic link for the genetic association between MAPT haplotypes and synucleinopathies.     

An antimicrobial protein of the Riptortus pedestris salivary gland was cleaved by a virulence factor of Serratia marcescens

Recently, our group demonstrated that the bean bug, Riptortus pedestris, is a good experimental symbiosis model to study the molecular cross-talk between the host insect and the gut symbiont. The Burkholderia symbiont is orally acquired by host nymphs from the environment in every generation. However, it is still unclear how Riptortus specifically interacts with entomopathogens that are abundant in the environmental soil. In preliminary experiments, we observed that a potent entomopathogen, Serratia marcescens, can colonize the midgut of Riptortus insects and was recovered from the midgut when Serratia cells were orally administered, suggesting that this pathogenic bacterium can escape host immune defenses in the salivary fluid. We examined how orally fed Serratia cells can survive in the presence of antimicrobial substances of the Riptortus salivary fluid. In this study, a 15 kDa trialysin-like protein from the salivary gland of R. pedestris and a potent virulence factor of Serratia cells, a serralysin metalloprotease, from the culture medium of S. marcescens were successfully purified to homogeneity. When the purified Riptortus trialysin (rip-trialysin) was incubated with purified serralysin, rip-trialysin was specifically hydrolyzed by serralysin, leading to the loss of antimicrobial activity. These results clearly demonstrated that a potent virulent metalloprotease of S. marcescens functions as a key player in the escape from the salivary fluid-mediated host immune response, resulting in successful colonization of S. marcescens in the host midgut.     

Concurrent infection with an intestinal helminth parasite impairs host resistance to enteric Citrobacter rodentium and enhances Citrobacter-induced colitis in mice

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To test the hypothesis that intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established by using the intestinal helminth Heligomosomoides polygyrus. To analyze the modulatory effect of a Th2-inducing helminth on the outcome of enteric bacterium Citrobacter rodentium infection, BALB/c and STAT 6 knockout (KO) mice were infected with H. polygyrus, C. rodentium, or both. We found that only BALB/c mice coinfected with H. polygyrus and C. rodentium displayed a marked morbidity and mortality. The enhanced susceptibility to C. rodentium and intestinal injury of coinfected BALB/c mice were shown to be associated with a significant increase in helminth-driven Th2 responses, mucosally and systemically, and correlated with a significant downregulation of protective gamma interferon and with a dramatic upregulation of the proinflammatory tumor necrosis factor alpha response. In addition, C. rodentium-associated colonic pathology in coinfected BALB/c mice was significantly enhanced, whereas bacterial burden was increased and clearance was delayed. In contrast, coinfection in STAT 6 KO mice failed to promote C. rodentium infection or to induce a more severe intestinal inflammation and tissue injury, demonstrating a mechanism by which helminth influences the development of host protective immunity and susceptibility to bacterial infections. We conclude that H. polygyrus coinfection can promote C. rodentium-associated disease and colitis through a STAT 6-mediated immune mechanism.     

Identification and characterization of a novel marine Bacillus cereus for mosquito control

Entomopathogenic bacteria to control mosquitoes are a promising environmentally friendly alternative to synthetic pesticides. In the present study, a novel mosquitocidal bacterium was isolated from marine soil collected from east coastal areas at Pondicherry (India). 16S rRNA gene sequence alignment depicted that this isolate belonged to Bacillus cereus VCRC-B520 (NCBI: KC-119192). Biochemical studies on bacterial growth, biomass, and toxin production have revealed that this strain could possibly be helpful in the production of a biopesticide in mosquito control. Toxicity assay with B. cereus against mosquito larvae has shown that the filariasis vector, Culex quinquefasciatus, is more susceptible than the other two species (Anopheles stephensi and Aedes aegypti). The LC₅₀ and LC₉₀ values for C. quinquefasciatus were 0.30 and 2.21 mg/L, respectively. No effect of B. cereus was found on nontargeted organisms. SDS-PAGE analysis and protein purification result from the cell mass of B. cereus have shown that a well-perceptible polypeptide was the dependable factor (85 kDa) for mosquitocidal action. Protein characterization (M/S MALDI-TOF) has shown that it is an endotoxin-specific insecticidal protein, namely “Cry4Aa”. Phylogenetic analyses of 16S rDNA gene sequence from this marine isolate have revealed the presence of homology among closely related Bacillus strains. Therefore, considerable interest has been shown on the identification of a potential mosquitocidal bacterium from marine environment (B. cereus), which was not reported earlier in view of the current scenario of the rapid development of resistance to Bacillus sphaericus in mosquito vector control program.     

Baculovirus-mediated expression of a Helicobacter pylori protein-based multiepitope hybrid gene induces a potent B cell response in mice

Helicobacter pylori is a gram-negative bacterium that is present in over half of the world's population. The colonization of the stomach?s gastric mucosa by H. pylori is related to the onset of chronic gastritis, peptic ulcer, and cancer. The estimated deaths from gastric cancer caused by this bacterial infection are in the 15,000–150,000 range. Current treatment for controlling the colonization of H. pylori includes the administration of two to four antibiotics and a gastric ATPase proton pump inhibitor. Nevertheless, the bacterium has shown increased resistance to antibiotics. Despite an extensive list of attempts to develop a vaccine, no approved vaccine against H. pylori is available. Recombinant viruses are a novel alternative for the control of primary pathogenic agents. In this work, we employed a baculovirus that carries a Thp1 transgene coding for nine H. pylori epitopes, some from the literature, and others were selected in silico from the sequence of H. pylori proteins (carbonic anhydrase, urease B subunit, gamma-glutamyl transpeptidase, Lpp20, Cag7, and CagL). We verified the expression of this hybrid multiepitopic protein in HeLa cells. Mice were inoculated with the recombinant baculovirus Bac-Thp1 using various administration routes: intranasal, intragastric, intramuscular, and a combination of intranasal and intragastric. We identified a strong adjuvant-independent IgG-antibody response in the serum of recombinant baculovirus-Thp1 inoculated mice, which was specific for a strain of H. pylori isolated from a human patient. The bacterium-specific IgG-antibodies were present in sera 125 days after the first vaccine administration. Also, H. pylori-specific IgA-antibodies were found in feces at 82 days after the first inoculation. A baculovirus-based vaccine for H. pylori is promising for controlling this pathogen in humans.     

Photorhabdus asymbiotica: Shedding Light on a Human Pathogenic Bioluminescent Bacterium

Photorhabdus asymbiotica is a gram-negative bacillus of the family Enterobacteriaceae. The bacterium displays the curious property of bioluminescence - the production of light without heat. It is recognized as a human pathogen in the United States and Australia, where it causes both locally invasive soft tissue infection and disseminated disease. In Australia, P. asymbiotica is vectored by the soil nematode Heterorhabditis gerrardi. The vector in the U.S. has not yet been identified, but it is likely to also be a Heterorhabditid nematode. Although it grows readily on conventional culture media, clinical microbiology laboratories may misidentify this pathogenic bacterium because of an over-reliance on commercial rapid identification systems.     

Tuf of Streptococcus pneumoniae is a surface displayed human complement regulator binding protein

Streptococcus pneumoniae is a Gram-positive bacterium, causing acute sinusitis, otitis media, and severe diseases such as pneumonia, bacteraemia, meningitis and sepsis. Here we identify elongation factor Tu (Tuf) as a new Factor H binding protein of S. pneumoniae. The surface protein PspC which also binds a series of other human immune inhibitors, was the first identified pneumococcal Factor H binding protein of S. pneumoniae. Pneumococcal Tuf, a 55 kDa pneumococcal moonlighting protein which is displayed on the surface of pneumococci, is also located in the cytoplasm and is detected in the culture supernatant. Tuf binds the human complement inhibitors Factor H, FHL-1, CFHR1 and also the proenzyme plasminogen. Factor H and FHL-1 bound to Tuf, retain their complement regulatory activities. Similarly, plasminogen bound to Tuf was accessible for the activator uPA and activated plasmin cleaved the synthetic chromogenic substrate S-2251 as well as the natural substrates fibrinogen and the complement proteins C3 and C3b. Taken together, Tuf of S. pneumoniae is a new multi-functional bacterial virulence factor that helps the pathogen in complement escape and likely also in ECM degradation.     

DLB and PDD: a role for mutations in dementia and Parkinson disease genes?

Based on the substantial overlap in clinical and pathological characteristics of dementia with Lewy bodies (DLB) and Parkinson disease with dementia (PDD) with Alzheimer disease (AD) and Parkinson disease (PD) we hypothesized that these disorders might share underlying genetic factors. The contribution of both sequence and copy number variants (CNVs) in known AD and PD genes to the genetic etiology of DLB and PDD however is currently unclear. Therefore, we performed a gene-based mutation analysis of all major AD and PD genes in 99 DLB and 75 PDD patients, including familial and sporadic forms, from Flanders, Belgium. Also, copy number variants in APP, SNCA, and PARK2 were determined. In the AD genes we detected proven pathogenic missense mutations in PSEN1 and PSEN2, and 2 novel missense variants in PSEN2 and MAPT. In the PD genes we identified 1 SNCA duplication, the LRRK2 R1441C founder mutation and 4 novel heterozygous missense variants with unknown pathogenicity. Our results suggest a contribution of established AD and PD genes to the genetic etiology of DLB and PDD though to a limited extent. They do support the hypothesis of a genetic overlap between members of the Lewy body disease spectrum, but additional genes still have to exist.     

NLRC4 expression in intestinal epithelial cells mediates protection against an enteric pathogen

The inflammasomes have an important role in connecting the detection of endogenous and microbial danger signals to caspase-1 activation and induction of protective immune responses. NLRC4 is a cytosolic NOD (nucleotide binding and oligomerization domain)-like receptor (NLR) that can trigger inflammasome formation in response to bacterial flagellin, an immunodominant antigen in the intestine. To characterize the role of NLRC4 in bacterially triggered intestinal inflammation, we used the murine pathogen Citrobacter rodentium, an extracellular, attaching/effacing bacterium similar to enterohemorrhagic Escherichia coli and enteropathogenic E. coli. Following infection with C. rodentium, we found that Nlrc4^−/− mice developed more severe weight loss, increased bacterial colonization levels, and exacerbated intestinal inflammation compared with wild-type counterparts. Nlrc4^−/− mice mounted robust adaptive immune responses but were unable to control early colonization by C. rodentium, suggesting that a defect in innate immunity was responsible. Experiments using bone marrow (BM) chimeras revealed that the protective effects of NLRC4 were dependent on its expression in non-hematopoietic cells, and quantitative PCR (Q-PCR) analyses revealed that NLRC4 was highly expressed in epithelial crypts but not in intestinal stroma. Thus, early NLRC4 sensing in intestinal epithelial cells regulates colonization by an extracellular bacterial pathogen and limits subsequent intestinal damage.     

Bacterial taxa associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR amplification and TTGE fingerprinting

Dermanyssus gallinae (Arthropoda, Mesostigmata) is suspected to be involved in the transmission of a wide variety of pathogens, but nothing is known about its associated non-pathogenic bacterial community. To address this question, we examined the composition of bacterial communities in D. gallinae collected from standard poultry farms in Brittany, France. Genetic fingerprints of bacterial communities were generated by temporal temperature gradient gel electrophoresis (TTGE) separation of individual polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments, followed by DNA sequence analysis. Most of the sequences belonged to the Proteobacteria and Firmicute phyla, with a majority of sequences corresponding to the Enterobacteriales order and the Staphylococcus genus. By using statistical analysis, we showed differences in biodiversity between poultry farms. We also determined the major phylotypes that compose the characteristic microbiota associated with D. gallinae. Saprophytes, opportunistic pathogens and pathogenic agents such as Pasteurella multocida, Erysipelothrix rhusiopathiae and sequences close to the genus Aerococcus were identified. Endosymbionts such as Schineria sp., Spiroplasma sp. Anistosticta, “Candidatus Cardinium hertigii” and Rickettsiella sp. were also present in the subdominant bacterial community. Identification of potential targets within the symbiont community may be considered in the future as a means of ectoparasite control.