Potential Virulence Determinants Research Articles

Comparative analysis of European subtype tick-borne encephalitis virus strains isolated from patients from Eastern Siberia and Eastern and Northern Europe

Background. Isolation of strains from tick-borne encephalitis (TBE) patients proved that the European subtype tick-borne encephalitis virus (TBEV-Eur) in Siberia is involved in regional human infectious pathology and causes a clinical picture similar to Western TBE. However, a comparative analysis of the genomes of TBEV-Eur strains isolated from TBE patients in Eastern Siberia and Europe has not been carried out.The aim. Genome comparative analysis and search for virulence determinants in TBEV-Eur strains isolated from patients in Eastern Siberia and Northern and Eastern Europe.Materials and methods. In current work, TBEV-Eur strain 1G-98 from the collection of Scientific Сentre for Family Health and Human Reproduction Problems (GenBank Acc. No. KY069119) was used. The analysis also included all complete genome sequences of TBEV-Eur strains from patients submitted in GenBank database at the time the study began. Complete genome sequencing of strain 1G-98 was performed using the Sanger method. The virulence of the strain was assessed by intracerebral and subcutaneous infection of laboratory mice.Results. It has been shown that, according to the coding region of the genomes, the level of differences between TBEV-Eur strains from Siberia does not exceed the previously established maximum for this subtype of 3.1 %. The strain 1G-98, isolated from the blood of TBE patient from the Irkutsk region demonstrated high cerebral and peripheral activity. In this strain, D67G mutation in E protein DII domain was revealed, which could potentially be associated with virulence, and a long deletion in the variable part of the 3’-noncoding genome region, comparable in length to the highly virulent strain Hypr from Europe was found.Conclusions. For the first time, a comparative analysis of the genomes of TBEV-Eur strains from TBE patients from the Asian part of Russia and Europe was carried out showing their genetic similarity, and potential virulence determinants were identified. Key words: tick-borne encephalitis virus, European subtype, genome, amino acid sequence, virulence determinants

Novel virulence determinants in VP1 regulate the assembly of enterovirus-A71.

EV-A71 causes hand, foot, and mouth disease in children. In this study, we discovered three highly conserved residues at positions 75, 78, and 88 of the capsid protein VP1 as the potential virulence determinants of EV-A71, which can influence viral replication by regulating the assembly of EV-A71. Mechanistic studies revealed that VP1-T75A could affect the maturation cleavage of the VP0 precursor, resulting in deficiencies in binding to the receptor SCARB2, viral attachment, internalization, and even uncoating. For the mutants of T78A and G88A, more noninfectious empty particles were produced during viral assembly. The discovery of these novel determinants of EV-A71 virulence will promote the study of the pathogenesis of enteroviruses.

Multiplex Determination of K-Antigen and Colanic Acid Capsule Variants of Cronobacter sakazakii

Cronobacter sakazakii is associated with the ingestion of contaminated reconstituted powdered infant formula (PIF), resulting in necrotizing enterocolitis, sepsis and meningitis in neonatal infants. Potential virulence determinants include the variable capsular polysaccharides; K-antigen and colanic acid (CA). Strains encoding for the capsule variant K2:CA2 have been strongly associated with neonatal meningitis cases. This study aimed to develop and apply a multiplex PCR assay to determine C. sakazakii K-antigen and colanic acid types. Twenty-six strains of C. sakazakii which had previously been isolated from food and environmental sources were used. These cover 18 multilocus sequence types and four serotypes. Based on our research findings, we have identified two K-antigen types present. Specifically, the K1-antigen was observed in sequence types ST1, ST8, ST20, ST23, ST64, ST198, ST263, ST264 and ST406, while the K2-antigen was present in ST4, ST9, ST12, ST13, ST136, ST233, ST245 and ST405. Additionally, we detected colanic acid (CA) type 1 in sequence types ST1, ST8, ST9, ST20, ST245 and ST405, and colanic acid (CA) type 2 in ST4, ST12, ST13, ST23, and ST64. We compared the predicted K-antigen and colanic acid types with the entire genome sequences of the strains. The comparison showed complete agreement between the PCR amplification results and the genomic analysis of the K-antigen and colanic acid-encoding regions. This assay is a useful tool for rapid identification of C. sakazakii, K-antigen and colanic acid types, in routine diagnoses and foodborne investigations. In addition, it will contribute to our knowledge of virulence factors associated with life-threatening neonatal meningitis.

HbpA from Glaesserella parasuis induces an inflammatory response in 3D4/21 cells by activating the MAPK and NF-κB signalling pathways and protects mice against G. parasuis when used as an immunogen

Glaesserella parasuis is usually a benign swine commensal in the upper respiratory tract, but virulent strains can cause systemic infection characterized by pneumonia, meningitis, and fibrinous polyserositis. The intensive pulmonary inflammatory response following G. parasuis infection is the main cause of lung injury and death in pigs. Vaccination has failed to control the disease due to the lack of extended cross-protection. Accumulating evidence indicates that the heme-binding protein A (HbpA) is a potential virulence determinant and a promising antigen candidate for the development of a broader range of vaccines. However, it is not yet known whether HbpA contributes to G. parasuis virulence or has any potential immune protective effects against G. parasuis. Here, we show that HbpA can induce the transcription and secretion of proinflammatory cytokines (IL-6, TNF-α, and MCP-1) in porcine alveolar macrophages (PAM, 3D4/31). The HbpA protein is recognized by Toll-like receptors 2 and 4 on 3D4/21 macrophages, resulting in the activation of MAP kinase and NF-κB signalling cascades and the transcription and secretion of proinflammatory cytokines. HbpA contributes to virulence and bacterial pulmonary colonization in C57BL/6 mice and plays a role in adhesion to host cells and evasion of the bactericidal effect of pulmonary macrophages. In addition, mice immunized with HbpA were partially protected against challenge by G. parasuis SC1401. The results suggest that HbpA plays an important role in the pathogenesis of disease caused by G. parasuis and lay a foundation for the development of a subunit or chimeric anti-G. parasuis vaccine.

Unveiling potential virulence determinants in Vibrio isolates from Anadara tuberculosa through whole genome analyses.

The genus Vibrio includes pathogenic bacteria able to cause disease in humans and aquatic organisms, leading to disease outbreaks and significant economic losses in the fishery industry. Despite much work on Vibrio in several marine organisms, no specific studies have been conducted on Anadara tuberculosa. This is a commercially important bivalve species, known as "piangua hembra," along Colombia's Pacific coast. Therefore, this study aimed to identify and characterize the genomes of Vibrio isolates obtained from A. tuberculosa. Bacterial isolates were obtained from 14 A. tuberculosa specimens collected from two locations along the Colombian Pacific coast, of which 17 strains were identified as Vibrio: V. parahaemolyticus (n = 12), V. alginolyticus (n = 3), V. fluvialis (n = 1), and V. natriegens (n = 1). Whole genome sequence of these isolates was done using Oxford Nanopore Technologies (ONT). The analysis revealed the presence of genes conferring resistance to β-lactams, tetracyclines, chloramphenicol, and macrolides, indicating potential resistance to these antimicrobial agents. Genes associated with virulence were also found, suggesting the potential pathogenicity of these Vibrio isolates, as well as genes for Type III Secretion Systems (T3SS) and Type VI Secretion Systems (T6SS), which play crucial roles in delivering virulence factors and in interbacterial competition. This study represents the first genomic analysis of bacteria within A. tuberculosa, shedding light on Vibrio genetic factors and contributing to a comprehensive understanding of the pathogenic potential of these Vibrio isolates.IMPORTANCEThis study presents the first comprehensive report on the whole genome analysis of Vibrio isolates obtained from Anadara tuberculosa, a bivalve species of great significance for social and economic matters on the Pacific coast of Colombia. Research findings have significant implications for the field, as they provide crucial information on the genetic factors and possible pathogenicity of Vibrio isolates associated with A. tuberculosa. The identification of antimicrobial resistance genes and virulence factors within these isolates emphasizes the potential risks they pose to both human and animal health. Furthermore, the presence of genes associated with Type III and Type VI Secretion Systems suggests their critical role in virulence and interbacterial competition. Understanding the genetic factors that contribute to Vibrio bacterial virulence and survival strategies within their ecological niche is of utmost importance for the effective prevention and management of diseases in aquaculture practices.

Detection and characterization of potential virulence determinants in Staphylococcus pseudintermedius and S. schleiferi strains isolated from canine otitis externa in Korea.

A recent increase in the occurrence of canine skin and soft tissue infections, including otitis externa and pyoderma, caused by antimicrobial-resistant Staphylococcus pseudintermedius and S. schleiferi has become a significant public and veterinary health issues. We investigated the virulence potentials associated with the occurrence of canine otitis externa in S. pseudintermedius and S. schleiferi. In this study, the prevalence of genes encoding leukocidins, exfoliative toxins, and staphylococcal enterotoxins (SEs) was investigated using previously characterized S. pseudintermedius (n = 26) and S. schleiferi (n = 19) isolates derived from canine otitis externa. Susceptibility to cathelicidins (K9CATH and PMAP-36) and hydrogen peroxide (H2O2) was also examined in both staphylococcal species. A high prevalence of genes encoding leukocidins (lukS/F-I, lukS1/F1-S, and lukS2/F2-S), exfoliative toxins (siet, expB, and sset), and SEs was identified in both S. pseudintermedius and S. schleiferi isolates. Notably, S. pseudintermedius isolates possessed higher number of SE genes, especially newer SE genes, than S. schleiferi isolates harboring egc clusters. Although no significant differences in susceptibility to K9CATH and H2O2 were observed between the two isolate groups, S. pseudintermedius isolates exhibited enhanced resistance to PMAP-36 compared to S. schleiferi isolates. These findings suggest that high a prevalence of various toxin genes together with enhanced resistance to cathelicidins may contribute to the pathogenicity of S. pseudintermedius and S. schleiferi in canine cutaneous infections.

The In Vitro Replication Cycle of Achromobacter xylosoxidans and Identification of Virulence Genes Associated with Cytotoxicity in Macrophages.

ABSTRACTAchromobacter xylosoxidans is an opportunistic pathogen implicated in a wide variety of human infections including the ability to colonize the lungs of cystic fibrosis (CF) patients. The role of A. xylosoxidans in human pathology remains controversial due to the lack of optimized in vitro and in vivo model systems to identify and test bacterial gene products that promote a pathological response. We have previously identified macrophages as a target host cell for A. xylosoxidans-induced cytotoxicity. By optimizing our macrophage infection model, we determined that A. xylosoxidans enters macrophages and can reside within a membrane bound vacuole for extended periods of time. Intracellular replication appears limited with cellular lysis preceding an enhanced, mainly extracellular replication cycle. Using our optimized in vitro model system along with transposon mutagenesis, we identified 163 genes that contribute to macrophage cytotoxicity. From this list, we characterized a giant RTX adhesin encoded downstream of a type one secretion system (T1SS) that mediates bacterial binding and entry into host macrophages, an important first step toward cellular toxicity and inflammation. The RTX adhesin is encoded by other human isolates and is recognized by antibodies present in serum isolated from CF patients colonized by A. xylosoxidans, indicating this virulence factor is produced and deployed in vivo. This study represents the first characterization of A. xylosoxidans replication during infection and identifies a variety of genes that may be linked to virulence and human pathology.IMPORTANCE Patients affected by CF develop chronic bacterial infections characterized by inflammatory exacerbations and tissue damage. Advancements in sequencing technologies have broadened the list of opportunistic pathogens colonizing the CF lung. A. xylosoxidans is increasingly recognized as an opportunistic pathogen in CF, yet our understanding of the bacterium as a contributor to human disease is limited. Genomic studies have identified potential virulence determinants in A. xylosoxidans isolates, but few have been mechanistically studied. Using our optimized in vitro cell model, we identified and characterized a bacterial adhesin that mediates binding and uptake by host macrophages leading to cytotoxicity. A subset of serum samples from CF patients contains antibodies that recognize the RTX adhesion, suggesting, for the first time, that this virulence determinant is produced in vivo. This work furthers our understanding of A. xylosoxidans virulence factors at a mechanistic level.

Characterization and Molecular Identification of Bacteria Isolated from Raw Camel’s Milk in Al-Ahsa, Saudi Arabia

Background: Camel’s milk has many benefits on human health, however, drinking fresh untreated camel’s milk may pose a serious health concern. Therefore, the current study aimed to determine the bacteriological quality of raw camel’s milk by isolating, characterizing and identifying bacterial strains from the untreated milk in Al-Ahsa region, Saudi Arabia. Methods: Three raw milk samples were collected under aseptic conditions and bacterial counts were determined using the serial dilution plate method. Discrete colonies were picked based on morphological differences. Five representative bacterial strains were characterized by phenotypic analysis, biochemical tests using the API 20E system and sequencing of 16S rRNA genes. Result: The results showed that the total bacterial counts in camel’s milk reached 3 × 107 CFU mL-1, which exceeds the limit specified by the Saudi Food and Drug Authority. The isolates formed colonies that were rounded, entire, convex and elevated from the surface of the agar. The diameter of the colonies ranged from 1 to 4 mm after a 48-h incubation at 30°C on the M17 medium. The isolates were able to metabolize 12 (60%) to 15 (75%) out of 20 different compounds as growth substrates present in the API 20E test system. Additionally, BLAST analysis of the 16S rRNA sequences of the five bacterial isolates revealed that they were closely related to various known bacterial genera (e.g., Bacillus, Kocuria and Pseudomonas), indicating the diverse composition of the microflora in raw camel’s milk. Unlike the other four bacterial isolates, a clear zone was noted around the growing one bacterial isolate, CMK1, highlighting a remarkable degradation of the hemoglobin in the erythrocytes, highlighting the potential existence virulence determinants. Good hygiene practices during milk production and handling are recommended to ensure high raw milk quality and avoid health risks. This study sheds light on the bacterial diversity of raw camel’s milk that has a direct impact on public health and the economy.

Comparative proteomic profiling reveals specific adaption of Vibrio anguillarum to oxidative stress, iron deprivation and humoral components of innate immunity

The gram-negative bacterium Vibrio (Listonella) anguillarum (VA) is the causative agent of vibriosis, a terminal hemorrhagic septicemia affecting the aquacultural industry across the globe. In the current study we used label-free quantitative proteomics to investigate how VA adapts to conditions that mimic defined aspects of vibriosis-related stress such as exposure to oxidative stress (H2O2), exposure to humoral factors of innate immunity through incubation with Atlantic salmon serum, and iron deprivation upon supplementation of 2,2′-dipyridyl (DIP) to the growth medium. We also investigated how regulation of virulence factors may be governed by the VA growth phase and availability of nutrients. All experimental conditions explored revealed stress-specific proteomic adaption of VA and only nine proteins were found to be commonly regulated in all conditions. A general observation made for all stress-related conditions was regulation of multiple metabolic pathways. Notably, iron deprivation and exposure to Atlantic salmon serum evoked upregulation of iron acquisition mechanisms. The findings made in the present study represent a source of potential virulence determinants that can be of use in the search for means to understand vibriosis. SignificanceVibriosis in fish and shellfish caused by V. anguillarum (VA) is responsible for large economic losses in the aquaculture sector across the globe. However, not much is known about the defense mechanism of this pathogen to percept and adapt to the imposed stresses during infection. Analyzing the response of VA to multiple host-related physiochemical stresses, the quantitative proteomic analysis of the present study indicates modulation of several virulence determinants and key defense networks of this pathogen. Our findings provide a theoretical basis to enhance our understanding of VA pathogenesis and can be employed to improve current intervention strategies to control vibriosis in aquaculture.

Whole genome analysis unveils genetic diversity and potential virulence determinants in Vibrio parahaemolyticus associated with disease outbreak among cultured Litopenaeus vannamei (Pacific white shrimp) in India

ABSTRACT Vibrio parahaemolyticus has caused widespread mortality in Indian shrimp aquaculture in recent years. However, there are insufficient genome data for the isolates from Indian shrimp vibriosis to analyze genetic diversity and track the acquisition of genetic features that could be involved in virulence and fitness. In this study, we have performed genome analysis of V. parahaemolyticus isolated from moribund shrimps collected from shrimp farms along coastal Karnataka, India, for better understanding of their diversity and virulence. Five newly sequenced genomes of V. parahaemolyticus along with 40 genomes retrieved from NCBI were subjected to comparative genome analysis. The sequenced genomes had an overall genome size of 5.2 Mb. MLST analysis and core genome phylogenomic analysis revealed considerable genetic diversity among the isolates obtained from the moribund shrimps. Interestingly, none of the V. parahaemolyticus isolates possessed the classical features (PirAB) of the strains associated with Acute Hepatopancreatic Necrosis Disease (AHPND). This study also revealed the presence of multiple virulence attributes, including ZOT, ACE and RTX toxins, secretion systems, and mobile genetic elements. The findings of this study provide insights into the possible transition of an environmental V. parahaemolyticus to emerge as pathogens of aquaculture species by increasing its virulence and host adaptation. Future studies focusing on continuous genomic surveillance of V. parahaemolyticus are required to study the evolution and transmission of new variants in shrimp aquaculture, as well as to design and implement biosecurity programs to prevent disease outbreaks.

Complete, closed and curated genome sequences of Photobacterium damselae subsp. piscicida isolates from Australia indicate mobilome-driven localized evolution and novel pathogenicity determinants.

Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the Vibrionaceae , often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen Photobacterium damselae subsp. piscicida isolated in Australia. These are also the first genome sequences of P. damselae subsp. piscicida isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian P. damselae subsp. piscicida are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in P. damselae subsp. piscicida , and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in P. damselae subsp. piscicida – a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene ssrA located on the main chromosome, homologous to a critical-to-virulence determinant in Yersinia pseudotuberculosis . Our study describes, to our knowledge, the only fully closed and manually curated genomes of P. damselae subsp. piscicida available to date, offering new insights into this important fish pathogen and its evolution.

Phenotypic and Genetic Evolutions of a Porcine Reproductive and Respiratory Syndrome Modified Live Vaccine after Limited Passages in Pigs.

Modified live vaccines (MLVs) against the porcine reproductive and respiratory syndrome virus (PRRSV) have been regularly associated with safety issues, such as reversion to virulence. In order to characterize the phenotypic and genetic evolution of the PRRSV-1 DV strain from the Porcilis® PRRS MLV after limited passages in pigs, three in vivo experiments were performed. Trial#1 aimed (i) at studying transmission of the vaccine strain from vaccinated to unvaccinated contact pigs. Trial#2 and Trial#3 were designed (ii) to assess the reproducibility of Trial#1, using another vaccine batch, and (iii) to compare the virulence levels of two DV strains isolated from vaccinated (passage one) and diseased contact pigs (passage two) from Trial#1. DV strain isolates from vaccinated and contact pigs from Trial#1 and Trial#2 were submitted to Next-Generation Sequencing (NGS) full-genome sequencing. All contact animals from Trial#1 were infected and showed significantly increased viremia compared to vaccinated pigs, whereas no such change was observed during Trial#2. In Trial#3, viremia and transmission were higher for inoculated pigs with passage two of the DV strain, compared with passage one. In this study, we showed that the re-adaptation of the DV strain to pigs is associated with faster replication and increased transmission of the vaccine strain. Punctually, a decrease of attenuation of the DV vaccine strain associated with clinical signs and increased viremia may occur after limited passages in pigs. Furthermore, we identified three mutations linked to pig re-adaptation and five other mutations as potential virulence determinants.

Comparative Secretome and Functional Analyses Reveal Glycoside Hydrolase Family 30 and Cysteine Peptidase as Virulence Determinants in the Pinewood Nematode Bursaphelenchus xylophilus.

Pine wilt disease, caused by the pinewood nematode, Bursaphelenchus xylophilus, is one of the world’s most serious tree diseases. Although the B. xylophilus whole-genome sequence and comprehensive secretome profile have been determined over the past decade, it remains unclear what molecules are critical in pine wilt disease and govern B. xylophilus virulence in host pine trees. Here, a comparative secretome analysis among four isolates of B. xylophilus with distinct virulence levels was performed to identify virulence determinants. The four candidate virulence determinants of B. xylophilus highly secreted in virulent isolates included lipase (Bx-lip1), glycoside hydrolase family 30 (Bx-GH30), and two C1A family cysteine peptidases (Bx-CAT1 and Bx-CAT2). To validate the quantitative differences in the four potential virulence determinants among virulence groups at the protein level, we used real-time reverse-transcription polymerase chain reaction analysis to investigate these determinants at the transcript level at three time points: pre-inoculation, 3 days after inoculation (dai), and 7 dai into pine seedlings. The transcript levels of Bx-CAT1, Bx-CAT2, and Bx-GH30 were significantly higher in virulent isolates than in avirulent isolates at pre-inoculation and 3 dai. A subsequent leaf-disk assay based on transient overexpression in Nicotiana benthamiana revealed that the GH30 candidate virulent factor caused cell death in the plant. Furthermore, we demonstrated that Bx-CAT2 was involved in nutrient uptake for fungal feeding via soaking-mediated RNA interference. These findings indicate that the secreted proteins Bx-GH30 and Bx-CAT2 contribute to B. xylophilus virulence in host pine trees and may be involved in pine wilt disease.

Screening Lactobacillus strains from artisanal Turkish goatskin casing Tulum cheeses produced by nomads via molecular and in vitro probiotic characteristics.

Eleven Lactobacillus (L.) strains were newly isolated from traditional Turkish Tulum cheeses and were characterized regarding their potential probiotic characteristics (bile and acid tolerance, gastric and pancreatic juice tolerance, lysozyme tolerance, adhesion ability), virulence determinants (hemolytic activity, antibiotic resistance, biogenic amine production), and functional properties (antibacterial activity, β-galactosidase activity, production of exopolysaccharides, cholesterol removal). These isolates were identified as L. brevis, L. plantarum, L. paracasei, L. coryniformis, L. rhamnosus and L. helveticus by 16S rRNA sequencing. With regard to safety aspects, none of the tested Lactobacillus isolates showed hemolytic activity or biogenic amine production. All the Lactobacillus isolates except isolate 24 were found to be sensitive or intermediate sensitive to penicillin, which is a frequently used antibiotic. Nine Lactobacillus isolates showed antibacterial activity against Staphylococcus aureus ATCC 25923, while only isolates 15 and 449 exhibited inhibitory activity against Listeria monocytogenes ATCC 7644. All isolated strains survived, even in the presence of 10.00 g L-1 bile after 48 h, and exhibited good survival at pH 3, but only two isolates survived at pH 2. Among the strains, isolate 15 exhibited satisfactory auto-aggregative, cell-surface hydrophobicity features, cholesterol-lowering activity and good acid tolerance. Isolate 15 also showed the strongest bile and simulated pancreatic juice resistance and moderate lysozyme tolerance. These outcomes suggest that isolate 15, identified as a L. plantarum strain from Tulum cheese, may be a promising probiotic candidate and could be suitable for use in several fermented foods. © 2020 Society of Chemical Industry.

The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection

BackgroundTick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection.MethodMice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated.ResultsWe identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains.ConclusionsOur findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.

The Serratia grimesii outer membrane vesicles-associated grimelysin triggers bacterial invasion of eukaryotic cells.

Serratia grimesii are facultative pathogenic bacteria that can penetrate a wide range of host cells and cause infection, especially in immunocompromised patients. Previously, we have found that bacterial metalloprotease grimelysin is a potential virulence determinant of S. grimesii invasion (E. S. Bozhokina et al., (2011).Cell Biology International, 35(2), 111-118). Protease is characterized as an actin-hydrolyzing enzyme with a narrow specificity toward other cell proteins. It is not known, however, whether grimelysin is transported into eukaryotic cells. Here, we show, for the first time, that S. grimesii can generate outer membrane vesicles (OMVs) displayed specific proteolytic activity against actin, characteristic of grimelysin. The presence of grimelysin was also confirmed by the Western blot analysis of S. grimesii OMVs lysate. Furthermore, confocal microscopy analysis revealed that the S. grimesii grimelysin-containing OMVs attached to the host cell membrane. Finally, pretreatment of HeLa cells with S. grimesii OMVs before the cells were infected with bacteria increased the bacterial penetration several times. These data strongly suggest that protease grimelysin promotes S. grimesii internalization by modifying bacterial and/or host molecule(s) when it is delivered as a component of OMVs.

Mapping Attenuation Determinants in Enterovirus-D68.

Enterovirus (EV)-D68 has been associated with epidemics in the United Sates in 2014, 2016 and 2018. This study aims to identify potential viral virulence determinants. We found that neonatal type I interferon receptor knockout mice are susceptible to EV-D68 infection via intraperitoneal inoculation and were able to recapitulate the paralysis process observed in human disease. Among the EV-D68 strains tested, strain US/MO-14-18949 caused no observable disease in this mouse model, whereas the other strains caused paralysis and death. Sequence analysis revealed several conserved genetic changes among these virus strains: nucleotide positions 107 and 648 in the 5′-untranslated region (UTR); amino acid position 88 in VP3; 1, 148, 282 and 283 in VP1; 22 in 2A; 47 in 3A. A series of chimeric and point-mutated infectious clones were constructed to identify viral elements responsible for the distinct virulence. A single amino acid change from isoleucine to valine at position 88 in VP3 attenuated neurovirulence by reducing virus replication in the brain and spinal cord of infected mice.

Inoculation With a Microbe Isolated From the Negev Desert Enhances Corn Growth.

Corn (Zea mays L.) is not only an important food source, but also has numerous uses, including for biofuels, fillers for cosmetics, glues, and so on. The amount of corn grown in the U.S. has significantly increased since the 1960’s and with it, the demand for synthetic fertilizers and pesticides/fungicides to enhance its production. However, the downside of the continuous use of these products, especially N and P fertilizers, has been an increase in N2O emissions and other greenhouse gases into the atmosphere as well as run-off into waterways that fuel pollution and algal blooms. These approaches to agriculture, especially if exacerbated by climate change, will result in decreased soil health as well as human health. We searched for microbes from arid, native environments that are not being used for agriculture because we reasoned that indigenous microbes from such soils could promote plant growth and help restore degraded soils. Employing cultivation-dependent methods to isolate bacteria from the Negev Desert in Israel, we tested the effects of several microbial isolates on corn in both greenhouse and small field studies. One strain, Dietzia cinnamea 55, originally identified as Planomicrobium chinense, significantly enhanced corn growth over the uninoculated control in both greenhouse and outside garden experiments. We sequenced and analyzed the genome of this bacterial species to elucidate some of the mechanisms whereby D. cinnamea 55 promoted plant growth. In addition, to ensure the biosafety of this previously unknown plant growth promoting bacterial (PGPB) strain as a potential bioinoculant, we tested the survival and growth of Caenorhabditis elegans and Galleria mellonella (two animal virulence tests) as well as plants in response to D. cinnamea 55 inoculation. We also looked for genes for potential virulence determinants as well as for growth promotion.

A ROD9 island encoded gene in Salmonella Enteritidis plays an important role in acid tolerance response and helps in systemic infection in mice

ABSTRACT Salmonella, like other pathogenic bacteria has undergone multiple genomic alterations to adapt itself into specific host environments executing varied degrees of virulence through evolution. Such variations in genome content have been assumed to lead the closely related non-typhoidal serovars, S. Enteritidis, and S. Typhimurium to exhibit Type Three Secretion System −2 (T3SS-2) based diverse colonization and inflammation kinetics. Mutually exclusive genes present in either of the serovars are recently being studied and in our currentwork, we focused on a particular island ROD9, present in S. Enteritidis but not in S. Typhimurium. Earlier reports have identified a few genes from this island to be responsible for virulence in vitro as well as in vivo. In this study, we have identified another gene, SEN1008 from the same island encoding a hypothetical protein to be a potential virulence determinant showing systemic attenuation upon mutation in C57BL/6 mice infection model. The isogenic mutant strain displayed reduced adhesion to epithelial cells in vitro as well as was highly immotile. It was also deficient in intracellular replication in vitro, with a highly suppressed SPI-2and failed to cause acute colitis at 72-h p.i.in vivo. Moreover, on acid exposure, SEN1008 showed 17 folds and 2 fold up-regulations during adaptation and challenge phases,respectively and ΔSEN1008 failed to survive during ATR assay, indicating its role under acid stress. Together, our findings suggested ΔSEN1008 to be significantly attenuated and we propose this gene to be a potent factor responsible for S. Enteritidis pathogenesis.

IpdE1-IpdE2 Is a Heterotetrameric Acyl Coenzyme A Dehydrogenase That Is Widely Distributed in Steroid-Degrading Bacteria.

Steroid-degrading bacteria, including Mycobacterium tuberculosis (Mtb), utilize an architecturally distinct subfamily of acyl coenzyme A dehydrogenases (ACADs) for steroid catabolism. These ACADs are α2β2 heterotetramers that are usually encoded by adjacent fadE-like genes. In mycobacteria, ipdE1 and ipdE2 (formerly fadE30 and fadE33) occur in divergently transcribed operons associated with the catabolism of 3aα-H-4α(3′-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), a steroid metabolite. In Mycobacterium smegmatis, ΔipdE1 and ΔipdE2 mutants had similar phenotypes, showing impaired growth on cholesterol and accumulating 5-OH HIP in the culture supernatant. Bioinformatic analyses revealed that IpdE1 and IpdE2 share many of the features of the α- and β-subunits, respectively, of heterotetrameric ACADs that are encoded by adjacent genes in many steroid-degrading proteobacteria. When coproduced in a rhodococcal strain, IpdE1 and IpdE2 of Mtb formed a complex that catalyzed the dehydrogenation of 5OH-HIP coenzyme A (5OH-HIP-CoA) to 5OH-3aα-H-4α(3′-prop-1-enoate)-7aβ-methylhexa-hydro-1,5-indanedione coenzyme A ((E)-5OH-HIPE-CoA). This corresponds to the initial step in the pathway that leads to degradation of steroid C and D rings via β-oxidation. Small-angle X-ray scattering revealed that the IpdE1-IpdE2 complex was an α2β2 heterotetramer typical of other ACADs involved in steroid catabolism. These results provide insight into an important class of steroid catabolic enzymes and a potential virulence determinant in Mtb.