Flagellin FLiC Enhances Resistance of Upland Cotton to Verticillium dahliae

The mechanism by which flagellin induces an immune response in plants is still unclear. The purpose of this study is to reveal the effect and mechanism of flagellin in inducing plants to produce an immune response to increase the resistance to Verticillium dahliae (VD). The full-length flagellin gene C (FliC) was cloned from an endophytic bacteria (Pseudomonas) in the root of upland cotton cultivar Zhongmiansuo 41. The FliC protein purified in vitro has 47.50% and 32.42% biocontrol effects on resistant and susceptible cotton cultivars, respectively. FLiC can induce allergic reactions in tobacco leaf cells and immune responses in cotton plants. Smearing FLiC to cotton and performing RNA-seq analysis, it is significantly enriched in the activity of positive ion transporters such as potassium ions and calcium ions (Ca2+), diterpenoid biosynthesis, phenylpropane biosynthesis and other disease-resistant metabolic pathways. FLiC inhibits the expression of calcium antiporter activity gene (GhCAA) to accelerate intracellular Ca2+ influx and stimulate the increase of intracellular hydrogen peroxide (H2O2) and nitric oxide (NO) content. The coordinated regulation of Ca2+, H2O2 and NO enhances disease resistance. The resistance of transgenic FLiC gene Arabidopsis to VD was significantly improved. The FLiC gene can be used as an anti-VD gene and as a regulator to improve resistance to VD.

Lack of N-terminal segment of the flagellin protein results in the production of a shortened polar flagellum in a deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913

Bacterial polar flagella, comprised of flagellin, are essential for bacterial motility. Pseudoalteromonas sp. SM9913 is a bacterium isolated from deep-sea sediments. Unlike other Pseudoalteromonas strains that have a long polar flagellum, strain SM9913 has an abnormally short polar flagellum. Here, we investigated the underlying reason for the short flagellar length and found that a single base mutation was responsible for the altered flagellar assembly. This mutation leads to the fragmentation of the flagellin gene into two genes, PSM_A2281 , encoding the core segment, and the C-terminal segment, and PSM_A2282 , encoding the N-terminal segment, and only gene PSM_A2281 is involved in the production of the short polar flagellum. When a chimeric gene of PSM_A2281 and PSM_A2282 encoding an intact flagellin A2281::82 was expressed, a long polar flagellum was produced, indicating that the N-terminal segment of flagellin contributes to the production of a polar flagellum of normal length. Analysis of the simulated structures of A2281 and A2281::82 and that of the flagellar filament assembled with A2281::82 indicates that, due to the lack of two α-helices, the core of the flagellar filament assembled with A2281 is incomplete, which is likely too weak to support the stability and movement of a long flagellum. This mutation in strain SM9913 had little effect on its growth and only a small effect on its swimming motility, implying that strain SM9913 can live well with this mutation in natural sedimentary environments. This study provides a better understanding of the assembly and production of bacterial flagella. Importance Polar flagella, which are an essential organelle for bacterial motility, are comprised of multiple flagellin subunits. A flagellin molecule contains an N-terminal segment, a core segment and a C-terminal segment. Results of this investigation of the deep-sea sedimentary bacterium Pseudoalteromonas sp. SM9913 demonstrate that a single base mutation in the flagellin gene leads to the production of an incomplete flagellin without the N-terminal segment and that the loss of the N-terminal segment of the flagellin protein results in the production of a shortened polar flagellar filament. Our results shed light on the important function of the N-terminal segment of flagellin in the assembly and stability of bacterial flagellar filament.

Long-term study of Borrelia and Babesia prevalence and co-infection in Ixodes ricinus and Dermacentor recticulatus ticks removed from humans in Poland, 2016–2019

Background Lyme borreliosis (LB) is the most common vector-borne disease in Europe. Monitoring changes in the prevalence of different Borrelia species in ticks may be an important indicator of risk assessment and of differences in pathogenicity in humans. The objective of our study was to assess the prevalence, co-infection and distribution of Borrelia and Babesia species in ticks removed from humans in a large sample collected during a study period of 4 years. Methods The ticks were collected throughout Poland from March to November over 4-year period from 2016 to 2019. All ticks (n = 1953) were morphologically identified in terms of species and developmental stage. Molecular screening for Borrelia and Babesia by amplification of the flagellin gene (flaB) or 18S rRNA marker was performed. Pathogen identity was confirmed by Sanger sequencing or PCR–restriction fragment length polymorphism analysis. Results The ticks removed from humans in Poland during this study belonged to two species: Ixodes ricinus (97%) and Dermacentor reticulatus (3%). High Borrelia prevalence (25.3%), including B. miyamotoi (8.4%), was confirmed in Ixodes ricinus ticks removed from humans, as was the change in frequency of occurrence of Borrelia species during the 4-year study. Despite Babesia prevalence being relatively low (1.3%), the majority of tested isolates are considered to be pathogenic to humans. Babesia infection was observed more frequently among Borrelia-positive ticks (2.7%) than among ticks uninfected with Borrelia (0.8%). The most frequent dual co-infections were between Borrelia afzelii and Babesia microti. The presence of Borrelia was also confirmed in D. reticulatus (12.7%); however the role of these ticks in spirochete transmission to susceptible hosts is still unclear. Conclusions Although the overall risk of developing LB after a tick bite is low in Europe, knowledge of the prevalence and distribution of Borrelia and Babesia species in ticks might be an important indicator of the risk of both these tick-borne diseases. Graphical abstract

Molecular Detection of Novel Borrelia Species, Candidatus Borrelia javanense, in Amblyomma javanense Ticks from Pangolins

A novel Borrelia species, Candidatus Borrelia javanense, was found in ectoparasite ticks, Amblyomma javanense, from Manis javanica pangolins seized in anti-smuggling operations in southern China. Overall, 12 tick samples in 227 (overall prevalence 5.3%) were positive for Candidatus B. javanense, 9 (5.1%) in 176 males, and 3 (5.9%) in 51 females. The phylogenetic analysis, based on the 16S rRNA gene and the flagellin gene sequences of the Borrelia sp., exhibited strong evidence that Candidatus B. javanense did not belong to the Lyme disease Borrelia group and the relapsing fever Borrelia group but another lineage of Borrelia. The discovery of the novel Borrelia species suggests that A. javanense may be the transmit vector, and the M. javanica pangolins should be considered a possible origin reservoir in the natural circulation of these new pathogens. To our knowledge, this is the first identification of a novel Borrelia species agent in A. javanense from pangolins. Whether the novel agent is pathogenic to humans is unknown and needs further research.

Selection and characterization of twoBacillus thuringiensisstrains showing nematicidal activity againstCaenorhabditis elegansandMeloidogyne incognita

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 μg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 μg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.

Ligilactobacillus agilis BKN88 possesses thermo-/acid-stable heteropolymeric flagellar filaments

Many flagellated bacteria possess multiple flagellins, but the roles and the compositions of each flagellin are diverse and poorly understood. In Ligilactobacillus agilis BKN88, there are two active flagellin gene paralogues but their function and composition in its flagellar filaments have not been described. The aim of this study is to find the function and composition of the flagellins by employing mutant strains each of which expresses a single flagellin or a modified flagellin. Two single flagellin-expressing strains were both flagellated while the number of flagella per cell in the single flagellin-expressing derivatives was lower than that in the wild type. Nonetheless, these derivative strains were apparently equally motile as the wild type. This indicates that either flagellin is sufficient for cell motility. The immunological activity via Toll-like receptor 5 of the single flagellin-expressing strains or purified single flagellins was readily detectable but mostly variably weaker than that of the wild type. The flagellar filaments of wild type L. agilis BKN88 were more acid-/thermo-stable than those of single flagellin-expressing derivatives. Using a combination of immunoprecipitation and flagellin-specific staining, wild type BKN88 appeared to possess heteropolymeric flagellar filaments consisting of both flagellins and each flagellin appeared to be equally distributed throughout the filaments. The results of this study suggest that the two flagellins together form a more robust filament than either alone and are thus functionally complementary.

Retrospective Survey of Borrelia spp. From Rodents and Ticks in Thailand

Borrelia is a genus of spirochetal bacteria with several species known to cause disease in humans. The distribution of Borrelia has rarely been studied in Thailand. In this study, a retrospective survey of Borrelia was conducted in ticks and wild rodents to better characterize the prevalence, diversity, and distribution of Borrelia across Thailand. Several pools of DNA from tick samples were positive for Borrelia spp. (36/258, 13.9%). Borrelia theileri/B. lonestari was found in 17 tick samples (16 pools of Haemaphysalis bandicota and 1 pool of Rhipicephalus sp.), and Borrelia yangtzensis was found in 8 tick samples (2 pools of H. bandicota and 6 pools of Ixodes granulatus). Borrelia spp. were detected at low prevalence levels in rodent tissue samples (24/2001, 1.2%), with 19 identified as B. theileri or B. lonestari and 5 identified as B. miyamotoi. Several geographic and species-specific infection trends were apparent, with Ixodes ticks infected with B. yangtzensis and Haemaphysalis and Rhipicephalus ticks infected with both B. yangtzensis and B. theileri/B. lonestari. Notably, B. yangtzensis showed a similar geographic distribution to B. miyamotoi, which was identified in new areas of Thailand in this study. The flagellin gene sequence from B. miyamotoi was more similar to European (99.3–99.9%) than Japanese (96.9–97.6%) genotypes. This study greatly expands the knowledge of Borrelia in Thailand and identified several Borrelia species for the first time. It also found several ticks and rodents infected with the pathogen that were not previously known to carry Borrelia.

Flagellar structures from the bacterium Caulobacter crescentus and implications for phage ϕCbK predation of multi-flagellin bacteria

ABSTRACTCaulobacter crescentus is a gram-negative alpha-proteobacterium that commonly lives in oligotrophic fresh and salt-water environments. C. crescentus is a host to many bacteriophages, including ϕCbK and ϕCbK-like bacteriophages, which first adsorb to cells by interaction with the bacterial flagellum. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate ϕCbK adsorption efficiency, although no single deletion completely ablates ϕCbK adsorption. Thus, bacteriophage ϕCbK likely recognizes a common motif amongst the six known flagellins in C. crescentus with varying degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by ϕCbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe post-translational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and ϕCbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 Å. Altogether, this work builds a platform for future investigation of how phage ϕCbK infects C. crescentus at the molecular level.

Emerging Threat of Salmonella typhimurium in Extraintestinal Infections: Cases from a Tertiary Care Hospital in North India

Purpose: Infections due to invasive non-typhoid salmonella can be dangerous and fatal. The mode of infection and the severity varies from the typhoidal fevers. It is important to find the association between clinical features and the infecting serovar to understand the pathophysiology and course of treatment. Methods: In the present study, extra-intestinal specimens (blood, cerebrospinal fluid and pus) from three patients suffering from septicaemia, meningitis and osteomyelitis were received. Micro-biological and biochemical test for species identification and antibiotic susceptibility was done as per standard protocol. Further, PCR based amplification and sequencing of a portion of the flagellin gene (FliC) was done to confirm the serovar. Results: Salmonella enterica was identified from all the three by microbiological and biochemical examination.The sequence of the Flic gene confirmed the serovar to be S. typhimurium. All the patients were treated successfully for the infection by appropriate antibiotic therapy. Conclusion: The study highlights that serovar Typhimurium is common in invasive non-typhoidal salmonellosis and its pathophysiology and virulence factors expression should be understood in various organ types for better treatment options and outcomes.