Pilus Protein Research Articles

Efficacy and Immune Correlates of OMP-1B and VirB2-4 Vaccines for Protection of Dogs from Tick Transmission of Ehrlichia chaffeensis.

Ehrlichia chaffeensis, an obligatory intracellular bacterium, causes human monocytic ehrlichiosis, an emerging disease transmitted by the Lone Star tick, Amblyomma americanum. Here, we investigated the vaccine potential of OMP-1B and VirB2-4. Among the highly expressed and immunodominant E. chaffeensis porin P28s/OMP-1s, OMP-1B is predominantly expressed by E. chaffeensis in A. americanum ticks, whereas VirB2-4 is a pilus protein of the type IV secretion system essential for E. chaffeensis infection of host cells. Immunization with recombinant OMP-1B (rOMP-1B) or recombinant VirB2-4 (rVirB2-4) protected mice from E. chaffeensis infection as effectively as Entry-triggering protein of Ehrlichia immunization. Dogs vaccinated with a nanoparticle vaccine composed of rOMP-1B or rVirB2-4 and an immunostimulating complex developed high antibody titers against the respective antigen. Upon challenge with E. chaffeensis-infected A. americanum ticks, E. chaffeensis was undetectable in the blood of rOMP-1B or rVirB2-4 immunized dogs on day 3 or 6 post-tick attachment and for the duration of the experiment, whereas dogs sham-vaccinated with the complex alone were persistently infected for the duration of the experiment. E. chaffeensis exponentially replicates in blood-feeding ticks to facilitate transmission. Previously infected ticks removed from OMP-1B-immunized dogs showed significantly lower bacterial load relative to ticks removed from sham-immunized dogs, suggesting in-tick neutralization. Peripheral blood leukocytes from rVirB2-4-vaccinated dogs secreted significantly elevated amounts of interferon-γ soon after tick attachment by ELISpot assay and reverse transcription-quantitative PCR, suggesting interferon-γ-mediated Ehrlichia inhibition. Thus, Ehrlichia surface-exposed proteins OMP-1B and VirB2-4 represent new potential vaccine candidates for blocking tick-borne ehrlichial transmission. IMPORTANCE Ehrlichia are tick-borne pathogens that cause a potentially fatal illness-ehrlichiosis-in animals and humans worldwide. Currently, no vaccine is available for ehrlichiosis, and treatment options are limited. Ticks are biological vectors of Ehrlichia, i.e., Ehrlichia exponentially replicates in blood-sucking ticks before infecting animals. Ticks also inoculate immunomodulatory substances into animals. Thus, it is important to study effects of candidate vaccines on Ehrlichia infection in both animals and ticks and the immune responses of animals shortly after infected tick challenge. Here, we investigated the efficacy of vaccination with functionality-defined two surface-exposed outer membrane proteins of Ehrlichia chaffeensis, OMP-1B and VirB2-4, in a mouse infection model and then in a dog-tick transmission model. Our results begin to fill gaps in our understanding of Ehrlichia-derived protective antigens against tick-transmission and immune correlates and mechanisms that could help future development of vaccines for immunization of humans and animals to counter tick-transmitted ehrlichiosis.

REACTION BETWEEN Shigella dysentriae SUBUNIT PILI ANTIGEN WITH 18 KDA Shigella flexneri SUBUNIT PILI ANTIBODY

Shigellosis caused by S. flexneri and S. dysentriae is the leading cause of death in developing countries. Meanwhile, the development of the Shigella vaccine is still ongoing. This study aims to prove the reaction between the 18 kDa S. flexneri pili sub-unit protein's antibodies and with the S. dysentriae pili sub-unit protein antigen. This study used the Western Blot and Dot Blot tests to determine the antibody-antigen reaction both qualitatively and quantitatively. From the study results, it was found that the 18 kDa S. flexneri 18 kDa pili subunit protein was able to recognize the S. dysentriae pili sub-unit protein with a molecular weight of 17 kDa and 23 kDa. The protein subunit pili S. dysentriae with a molecular weight of 17 kDa and 23 kDa probably has an epitope recognized by the antibody to the pili protein subunit S. flexneri 18 kDa. The equivalent zone of the antibody-antigen reaction lies in the concentration of the antibody protein subunit pili 18 kDa S. flexneri titer 1/300 and the concentration of the antigen protein subunit pili 18 kDa S. dysentriae titer 1/20. So that the higher the dilution of the antibody of the 18 kDa S. flexneri pili subunit, the lower the bond with the S. dysentriae pili subunit antigen.

Pili Protein 65.5 kDa of Klebsiella pneumoniae Induced a Decrease in IL-10 in Mice

Klebsiella pneumoniae is one of the leading cause of nosocomial infection worldwide with clinical isolates mostly found in Multi-Drug Resistant (MDR) or Extensively Drug-Resistant (XDR). This makes therapeutic options due to this bacteria limited. Several studies have shown that the pili protein of K. pneumoniae with different molecular weights plays a role in the virulence factor, as they are hemagglutinin and adhesin proteins. Pili protein 65.5 kDa of K. pneumoniae can be considered as the antigen candidate for vaccines. This study aimed to determine the immune response based on the IL-10 level as induced by pili protein 65.5 kDa K. pneumoniae. This study was conducted from October 2020 to March 2021 at the Faculty of Mathematics and Natural Sciences and Faculty of Medicine of Jember University. This was true experimental with post-test only controlled group design on 27 male BALB/c mice which divided into 3 groups: K1, K2, and K3. The groups were induced with sterile PBS, pili protein and Freund’s adjuvant, and Freund’s adjuvant only, respectively. Samples were drawn through cardiac puncture, then serum was taken to measure the IL-10 levels using the ELISA method. Hemagglutination test showed a positive result with 1/8 as the highest titer. Results showed that the mean IL-10 levels were 290.92±45.33; 235.05±44.53; 218.54±64,81 for K1, K2, and K3, respectively. One-way ANOVA test results showed a statistical difference between groups (p=0,019). This research shows that pili protein 65.5 kDa K. pneumoniae decreases the IL-10 level. However, when it is provided with the Freund’s Adjuvant, there is no proof of the immunogenicity when compared to the control group.

Characterization of Pili Protein 67 kDa Streptococcus pneumoniae: New Candidate for Virulence Factor-Based Pneumococcal Antigen Vaccine.

Streptococcus pneumoniae is a Gram-positive diplococci bacteria that causes infectious diseases such as otitis, meningitis, and pneumonia. Streptococcus pneumoniae has various virulence factors, one of which is pilus. In addition to being immunogenic, pilus S. pneumoniae also plays a role in bacterial adhesion to host cells and biofilm formation. The S. pneumoniae pilus found in this study consisted of several proteins with various molecular weights, one of which was a 67 kDa protein. This study aimed to determine the characteristics of the 67 kDa pilus protein, including its capacity as hemagglutinin and adhesin and its amino acid sequence (AA). The LCMS/MS method is used to determine the AA sequence of the 67 kDa pilus protein. The AA structure was analyzed through BLASTP by matching it with the sequence of the protein data bank of S. pneumoniae (taxid: 1313). The ProtParam tool from ExPASY was used to calculate various physical and chemical parameters of the protein, while for evaluating its immunogenicity, the VaxiJen V2.0 online server was used. The results of this study indicate that the 67 kD a pilus protein, is an anti-hemagglutinin protein and has a role as an adhesin protein. Adhesion tests show the action between protein concentration and the number of bacteria attached to enterocyte cells. LCMS/MS test results obtained by BLASTP showed that the 67 kDa pilus protein had three AA sequences (ITYMSPDFAAPTLAGLDDATK, AEFVEVTK, and LVVSTQTALA), which had similarities with the A backbone chain of S. pneumoniae pilus. The physicochemical test showed that the protein is hydrophilic and nonpolar, while the antigenicity test showed that the protein is antigenic. Based on these characteristics, it can be concluded that the 67 kDa S. pneumoniae pilus protein can be used as a vaccine candidate for pneumococcus.

A Five Year Randomized Controlled Trial to Assess the Efficacy and Antibody Responses to a Commercial and Autogenous Vaccine for the Prevention of Infectious Bovine Keratoconjunctivitis.

A randomized control trial was performed over a five-year period to assess the efficacy and antibody response induced by autogenous and commercial vaccine formulations against infectious bovine keratoconjunctivitis (IBK). Calves were randomly assigned each year to one of three arms: an autogenous vaccine treatment that included Moraxella bovis (M. bovis), Moraxella bovoculi, and Mycoplasma bovoculi antigens, a commercial M. bovis vaccine treatment, or a sham vaccine treatment that consisted only of adjuvant. A total of 1198 calves were enrolled in the study. Calves were administered the respective vaccines approximately 21 days apart, just prior to turnout on summer pastures. Treatment effects were analyzed for IBK incidence, retreatment incidence, 205-day adjusted weaning weights, and antibody response to the type IV pilus protein (pili) of M. bovis as measured by a novel indirect enzyme-linked immunosorbent screening assay (ELISA). Calves vaccinated with the autogenous formulation experienced a decreased cumulative incidence of IBK over the entire study compared to those vaccinated with the commercial and sham formulations (24.5% vs. 30.06% vs. 30.3%, respectively, p = 0.25), and had less IBK cases that required retreatment compared to the commercial and sham formulations (21.4% vs. 27.9% vs. 34.3%, respectively, p = 0.15), but these differences were not significant. The autogenous formulation induced a significantly stronger antibody response than the commercial (p = 0.022) and sham formulations (p = 0.001), but antibody levels were not significantly correlated with IBK protection (p = 0.37).

Multiple Proteins of Lacticaseibacillus rhamnosus GG Are Involved in the Protection of Keratinocytes From the Toxic Effects of Staphylococcus aureus

We have previously shown that lysates of Lacticaseibacillus rhamnosus GG confer protection to human keratinocytes against Staphylococcus aureus. L. rhamnosus GG inhibits the growth of S. aureus as well as competitively excludes and displaces the pathogen from keratinocytes. In this study, we have specifically investigated the anti-adhesive action. We have tested the hypothesis that this activity is due to quenching of S. aureus binding sites on keratinocytes by molecules within the Lacticaseibacillus lysate. Trypsinisation or heat treatment removed the protective effect of the lysate suggesting the involvement of proteins as effector molecules. Column separation of the lysate and analysis of discrete fractions in adhesion assays identified a fraction of moderate hydrophobicity that possessed all anti-adhesive functions. Immunoblotting demonstrated that this fraction contained the pilus protein, SpaC. Recombinant SpaC inhibited staphylococcal adhesion to keratinocytes in a dose-dependent manner and improved keratinocyte viability following challenge with viable S. aureus. However, SpaC did not confer the full anti-adhesive effects of the LGG lysate and excluded but did not displace S. aureus from keratinocytes. Further purification produced four protein-containing peaks (F1–F4). Of these, F4, which had the greatest column retention time, was the most efficacious in anti-staphylococcal adhesion and keratinocyte viability assays. Identification of proteins by mass spectrometry showed F4 to contain several known “moonlighting proteins”—i.e., with additional activities to the canonical function, including enolase, Triosephosphate isomerase (TPI), Glyceraldehyde 3 phosphate dehydrogenase (G3P) and Elongation factor TU (EF-Tu). Of these, only enolase and TPI inhibited S. aureus adhesion and protected keratinocytes viability in a dose-dependent manner. These data suggest that inhibition of staphylococcal binding by the L. rhamnosus GG lysate is mediated by SpaC and specific moonlight proteins.

A type IVB pilin influences twitching motility and in vitro adhesion to epithelial cells in Burkholderia pseudomallei.

Type IV pili are involved in adhesion, twitching motility, aggregation, biofilm formation and virulence in a variety of Gram-negative bacteria. Burkholderia pseudomallei, the causative agent of melioidosis and a Tier 1 biological select agent, is a Gram-negative bacterium with eight type IV pili-associated loci (TFP1 to TFP8). Most have not been fully characterized. In this study, we investigated BPSS2185, an uncharacterized TFP8 gene that encodes a type IVB pilus protein subunit. Using genetic deletion and complementation analysis in B. pseudomallei JW270, we demonstrate that BPSS2185 plays an important role in twitching motility and adhesion to A549 human alveolar epithelial cells. Compared to JW270, the JW270 ΔBPSS2185 mutant failed to display twitching motility and did not adhere to the epithelial cells. These phenotypes were partially reversed by the complementation of BPSS2185 in the mutant strain. The study also shows that BPSS2185 is expressed only during the onset of mature biofilm formation and at the dispersal of a biofilm, suggesting that the motility characteristic is required to form a biofilm. Our study is the first to suggest that the BPSS2185 gene in TFP8 contributes to twitching motility, adhesion and biofilm formation, indicating that the gene may contribute to B. pseudomallei virulence.

Pilus proteins from Streptococcus pyogenes stimulate innate immune responses through Toll-like receptor 2.

The group A Streptococcus (GAS) pilus is a long, flexible, hair‐like structure anchored to the cell surface that facilitates the adherence of GAS to host cells, thus playing a critical role in initiating infections. Because of its important role in GAS virulence, the pilus has become an attractive target for vaccine development. While current research mainly focuses on pilus function and its potential as a vaccine component, there is a lack of knowledge on how the host immune system recognizes and responds to this abundant surface structure. Here we show that both assembled GAS pili and individual pilus proteins induce a potent release of the proinflammatory cytokines tumor necrosis factor and interleukin‐8. We further show that the surface‐exposed backbone pilin and ancillary pilin 1 subunits are Toll‐like receptor 2 (TLR2) agonists. Using reporter cell lines coexpressing human TLR2 in combination with either TLR1 or TLR6, we determined that activation was mediated by the TLR2/TLR6 heterodimer. Finally, we used solid‐phase and flow cytometry binding assays to illustrate a direct interaction between the pilus subunits and TLR2. These results provide further support for the suitability of the pilus as a vaccine component and opens potential avenues for using GAS pili as an adjuvant or immune‐modulation agent.

Conjugative antibiotic-resistant plasmids promote bacterial colonization of microplastics in water environments

Both microplastic and bacterial antibiotic resistance have attracted attention worldwide. When microplastics coexist with antibiotic-resistant bacteria (ARB), which carry antibiotic resistance genes (ARGs), ARB colonize the surface of microplastics, and a unique biofilm is formed. The ARB and ARGs in biofilms are denser and more difficult to remove. However, studies on the factors influencing the formation of microplastic biofilms are limited. In this study, plasmid RP4, which appeared in wastewater treatment plants, was found to be able to promote irreversible bacterial colonization of microplastics, and the hypothetical reason was conjugative pili expression. Then, the potential conjugative pili synthesis promoter “nanoalumina” and inhibitor “free nitrous acid” (FNA) were selected to test this hypothesis. Simultaneously, nanoalumina promoted and FNA inhibited bacterial colonization when RP4 existed. Combined with the gene expression and ATP analysis results, this hypothesis was confirmed, and the mechanism of RP4 on bacterial colonization was related mainly to conjugative pili protein synthesis and intracellular ATP. In this study, the effects of plasmid RP4, nanoalumina, and FNA on the formation of microplastic biofilms were reported, which has a certain reference value for other researchers exploring microplastic biofilms.

Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever

Abstract. Darmawati S, Ethica SN, Prastiyanto ME, Depamede SN, Putri EO, Kamaruddin M. 2022. Molecular characterization of a 42 kDa subunit pili protein of Salmonella typhi causes typhoid fever. Biodiversitas 23: 962-968. Blood culture is the gold standard for diagnosing typhoid fever, but it has limitations such as media and laboratory equipment, specimen volume, and examination time. However, the Academy of Pediatrics does not recommend serology due to its low sensitivity.The purpose of this study was to determine the molecular properties of the protein pilli of Salmonella typhi(S. typhi) that the findings can be used to develop a typhoid fever diagnostic reagent. The SDS-PAGE method was used, as well sequence analysis with ProtParam, ProtScale, and PSIPRED. The SDS-PAGE profile reveals one major protein (42 kDa) and fourteen minor proteins. The pili protein subunit 42 kDa had an amino acid (AA) sequence with a length of 390 AA, according to bioinformatics analysis. According to the ProtParam results, the pili protein subunit 42 kDa has good stability with a value of 40 and is a hydrophilic protein with an average GRAVY value of -0.950. PSIPRED results show that among the secondary structural elements, coil strand predominates, followed by -helix and -strand. It is concluded that this protein is immunogenic and that it can be used to develop a more specific and sensitive diagnostic reagent for typhoid fever.

Shigella flexneri vaccine development: Oral administration of peptides derived from the 49.8 kDa pili protein subunit activates the intestinal immune response in mice.

Background and Aim:The morbidity and mortality of Shigella infections remain a global challenge. Epitope-based vaccine development is an emerging strategy to prevent bacterial invasion. This study aimed to identify the ability of the 49.8 kDa pili subunit adhesin protein epitope of Shigella flexneri to induce an intestinal immune response in mice.Materials and Methods:Thirty adult male Balb/c mice were divided into a control group, cholera toxin B subunit (CTB) group, CTB+QSSTGTNSQSDLDS (pep_1) group, CTB+DTTITKAETKTVTKNQVVDTPVTTDAAK (pep_2) group, and CTB+ ATLGATLNRLDFNVNNK (pep_3). We performed immunization by orally administering 50 μg of antigen and 50 μl of adjuvant once a week over 4 weeks. We assessed the cellular immune response by quantifying T helper 2 (Th2) and Th17 using flow cytometry. In addition, we assessed the humoral immune response by quantifying interleukin (IL-4), IL-17, secretory immunoglobulin A (sIgA), and β-defensin using enzyme-linked immunoassay. Statistical analysis was performed using one-way analysis of variance and Kruskal–Wallis test.Results:Peptide oral immunization increases the cellular immune response as reflected by the increase of Th2 (p=0.019) and Th17 (p=0.004) cell counts, particularly in the CTB_pep_1 group. Humoral immune response activation was demonstrated by increased IL-4 levels, especially in the CTB+pep_3 group (p=0.000). The IL-17 level was increased significantly in the CTB+pep_1 group (p=0.042). The mucosal immune response was demonstrated by the sIgA levels increase in the CTB+pep_3 group (p=0.042) and the β-defensin protein levels (p=0.000).Conclusion:All selected peptides activated the cellular and humoral immune responses in the intestine of mice. Further studies are necessary to optimize antigen delivery and evaluate whether the neutralizing properties of these peptides allow them to prevent bacterial infection.

Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces—predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.

Construction of the Enterococcal Strain Expressing Immunogenic Fragment of SARS-Cov-2 Virus

Contemporary SARS-Cov-2 pandemic, besides its dramatic global influence on the human race including health care systems, economies, and political decisions, opened a window for the global experiment with human vaccination employing novel injectable vaccines providing predominantly specific IgG response with little knowledge of their impact on the mucosal immunity. However, it is widely accepted that protection against the pathogens at the gates of the infection - on mucosal surfaces—predominantly rely on an IgA response. Some genetically modified bacteria, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Probiotic-based vaccines for mucous membranes are easy to produce in large quantities; they have low cost, provide quite a long T-cell memory, and gut IgA response to oral vaccines is highly synchronized and strongly oligoclonal. Here we present a study demonstrating construction of the novel SARS-Cov-2 vaccine candidate employing the gene fragment of S1 SARS-Cov-2 gene. This DNA fragment was inserted in frame into major pili protein gene with d2 domain of enterococcal operon encoding for pili. The DNA sequencing proved the presence of the insert in enterococcal genome. RNA transcription, immunoprecipitation, and immune electron microscopy with human sera obtained from the SARS-Cov-2 patients demonstrated expression of SARS-Cov-2 antigens in bacteria. Taken together the data obtained allowed considering this genetically modified probiotic strain as an interesting candidate for vaccine against SARS-Cov-2.

Genomic investigation of a Streptococcus pneumoniae serotype 24F strain causing meningoencephalitis in Hong Kong

Pneumococcal conjugate vaccines (PCVs) successfully decreased the incidence of invasive pneumococcal disease in children. However, many countries have reported serotype replacement and a rebound in diseases from non-vaccine serotypes. Here, we report the genomic investigation of a Streptococcus pneumoniae strain M215 that caused severe meningoencephalitis in an infant in 2019. The strain was assigned to serotype 24F using the bioinformatic pipeline SeroBA and pneumococcal type specific anti-sera. The strain was resistant to cotrimoxazole from mutations in both folA and folP genes. It was susceptible to penicillin and other non-β-lactam antibiotics. Phylogenetically, it belongs to Global Pneumococcal Sequence Cluster (GPSC) 6 and multi-locus sequence type 162. A total of 38 virulence genes were detected in the genome of M215. Upon comparison of the profile of virulence genes, GPSC6 but not non-GPSC6 strains of serotype 24F and related serotypes were found to possess the major virulence determinant, pilus islet-1, comprising genes encoding sortases (srtB, srtC, srtD), pilus proteins (rrgA, rrgB and rrgC) and one transcriptional regulator (rlrA), which was previously described to be characteristic feature of international clones in the pre-PCV era. In our locality, this represented the first detection of serotype 24F and GPSC6/ST162 causing serious pneumococcal disease. The emergence of the non-vaccine serotype 24F GPSC6/ST162 lineage with molecular feature of high virulence is concerning and emphasizes the need for full characterization of strains causing severe disease.

LrpCBA pilus proteins of gut-dwelling Ligilactobacillus ruminis: crystallization and X-ray diffraction analysis.

Adhesion to host surfaces for bacterial survival and colonization involves a variety of molecular mechanisms. Ligilactobacillus ruminis, a strict anaerobe and gut autochthonous (indigenous) commensal, relies on sortase-dependent pili (LrpCBA) for adherence to the intestinal inner walls, thereby withstanding luminal content flow. Here, the LrpCBA pilus is a promiscuous binder to gut collagen, fibronectin and epithelial cells. Structurally, the LrpCBA pilus displays a representative hetero-oligomeric arrangement and consists of three types of pilin subunit, each with its own location and function, i.e. tip LrpC for adhesion, basal LrpB for anchoring and backbone LrpA for length. To provide further structural insights into the assembly, anchoring and functional mechanisms of sortase-dependent pili, each of the L. ruminis pilus proteins was produced recombinantly for crystallization and X-ray diffraction analysis. Crystals of LrpC, LrpB, LrpA and truncated LrpA generated by limited proteolysis were obtained and diffracted to resolutions of 3.0, 1.5, 2.2 and 1.4 Å, respectively. Anomalous data were also collected from crystals of selenomethionine-substituted LrpC and an iodide derivative of truncated LrpA. Successful strategies for protein production, crystallization and derivatization are reported.

Establishing an invertebrate Galleria mellonella greater wax moth larval model of Neisseria gonorrhoeae infection

ABSTRACT Neisseria gonorrhoeae (gonococcus) causes the human sexually transmitted disease gonorrhea. Studying gonococcal pathogenesis and developing new vaccines and therapies to combat the increasing prevalence of multi-antibiotic resistant bacteria has made use of many ex vivo models based on human cells and tissues, and in vivo vertebrate models, for example, rodent, pig and human. The focus of the current study was to examine the utility of the invertebrate greater wax moth Galleria mellonella as an in vivo model of gonococcal infection. We observed that a threshold of ~106 – 107 gonococci/larva was required to kill >50% of larvae (P < 0.05), and increased toxicity correlated with reduced health index scores and pronounced histopathological changes such as increases in the total lesion grade, melanized nodules, hemocyte reaction, and multifocal adipose body degeneration. Larval death was independent of the expression of pilus or Opa protein or LOS sialylation within a single gonococcal species studied, but the model could demonstrate relative toxicity of different isolates. N. meningitidis, N. lacatamica and gonococci all killed larvae equally, but were significantly less toxic (P > 0.05) than Pseudomonas aeruginosa. Larvae primed with nontoxic doses of gonococci were more susceptible to subsequent challenge with homologous and heterologous bacteria, and larval survival was significantly reduced (P < 0.05) in infected larvae after depletion of their hemocytes with clodronate-liposomes. The model was used to test the anti-gonococcal properties of antibiotics and novel antimicrobials. Ceftriaxone (P < 0.05) protected larvae from infection with different gonococcal isolates, but not azithromycin or monocaprin or ligand-coated silver nanoclusters (P > 0.05).

Atomic and Electronic Structure of Pilus from Geobacter sulfurreducens through QM/MM Calculations: Evidence for Hole Transfer in Aromatic Residues.

Long-range electron transport has been widely and experimentally reported in Geobacter sulfurreducens pilus protein. However, a better understanding of the still undefined molecular arrangement can bring to light the role of key residues in this phenomenon. We propose a theoretical investigation of the electronic structure of aromatic residue groups in the protein through a classical molecular dynamics (MD) simulation, followed by a quantum mechanics/molecular mechanics (QM/MM) electronic study of different frames sampled from MD trajectories, an electrostatic potential and electron density analysis, an analysis of the density of states, and an investigation of hole formation through Dyson orbital calculations. We observe a highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy gap in the ranges of 1.4-2.3 eV and 2.9-3.3 eV and a less intense dipole moment along the aromatic residues in the presence of water in comparison to the system in vacuum. HOMO and LUMO electron densities highlight the occupation of one tyrosine residue in every representation for HOMO and a delocalization along two to three rings for LUMO. The results show how the electronic structure of the aromatic residues is sensitive to the ring arrangement and the surrounding environment. In our study, we observe that slight rearrangements in the fiber geometry can create temporary conditions for hole transfer.

Metatranscriptome Analysis of Bisphenol A-Exposed Aerobic Granular Sludge

Understanding of the metabolic pathways connected with a removal of micropollutant bisphenol A (BPA) may help to better design effective wastewater treatment processes. The aim of this study was to determine changes in gene expression in an aerobic granular sludge (AGS) community exposed to BPA. In the study, AGS adapted to BPA degradation was used. In this sludge, BPA was dosed; as a control sample, granules without BPA addition were used. mRNA was isolated from both samples and sequenced using the Illumina platform. Metatranscriptome analysis of AGS exposed to BPA indicated direct biodegradation as the main mechanism of BPA removal from wastewater. High expression of genes coding pilus and flagellin proteins in the BPA-exposed biomass indicated that exposition to BPA stimulated aggregation of microbial cells and formation of biofilm. In the BPA-exposed biomass, nitrogen was mainly used as an energy source, as indicated by the presence of genes coding nitrification enzymes and urease. Moreover, exposition to BPA stimulated expression of genes coding proteins responsible for xenobiotic degradation, including enzymes responsible for benzoate degradation. These results increase knowledge about BPA metabolism in complex microbial communities in wastewater treatment systems and indicate that AGS is suitable for efficiently removing BPA from wastewater.

The Effect of Hibiscus Flower Extract (Hibiscus rosa-sinensis L.) on the Growth of Streptococcus sanguinis Bacteria

According to the data of Global Burden Disease in 2016, periodontal disease is the 11th disease suffered by 750,487 people worldwide. Gingivitis occurs due to dental plaque formation. A half of bacterial plaque populations are Streptococcus sp. Streptococcus sanguinis is a predominant bacterial that initiates plaque formation. The adherence of this bacteria is mediated by fimbriae, pilus protein, lipoprotein, and glucosyltransferase enzymes. Hibiscus flower has a potential as an antimicrobial agent that may inhibit plaque formation due its active component i.e. flavonoid, tannins, and saponin. This study aimed to investigate the effect of Hibiscus rosa-sinensis L. flower extract concentration on the growth of Streptococcus sanguinis. Wells diffusion method was used in this study, using 100 μl bacterial suspension of 1,5 x 108 CFU/ml of bacteria. Hibiscus flower extract (2%, 4%, 6%), chlorhexidine 0,12% as a positive control, and distilled water as a negative control were added in the agar wells. The results showed that all concentrations (2 %, 4%, 6 %) of Hibiscus flower extract higher significantly compared to negative control. The concentration of 4% and 6% are higher ;significantly compared to positive control. The largest inhibitory zone was at a concentration of 6% (6,35±0,09 mm) and the smallest inhibitory zone was at a concentration of 2% (2,51±0,12 mm). The conclusion is that the concentration of Hibiscus rosa-sinensis L. flower extract affects the growth of Streptococcus sanguinis.

Characterization of pili protein with molecular mass of 85 kDa Escherichia coli as an adhesin and a hemagglutinin

Background: Escherichia coli (E. coli) is a rod-shaped gram-negative bacterium included in Multi Drug Resistant (MDR) bacteria. There are several strategies in place to prevent and treat E. coli bacterial infections to reduce some the incidences of MDR. One of them is the development of a protein-based vaccine. Pili is one of virulence factors in E. coli surface proteins that can mediate attachments of bacteria to host cells (adhesin or hemagglutinin) and can be used as protein-based vaccine candidates. Objective: This study aims to determine characterization of pili protein with molecular mass of 85 kDa Escherichia coli bacteria as an adhesin and a hemagglutinin. Methods: This study is a true experimental laboratories and a descriptive study to determine roles of pili protein with molecular mass of 85 kDa as an adhesin and a hemagglutinin. Samples used in this study were a stock of E. coli in the Microbiology Laboratory Faculty of Medicine, Universitas Negeri Jember (UNEJ). A hemagglutination test in this study used mouse erythrocytes while an adhesion test used mouse enterocytes. Isolation and purification of E. coli pili protein, isolation of mouse erythrocytes, isolation of mouse enterocytes, the hemagglutination test and the adhesion test were methods in this study. A data analysis with a correlation-regression was used to determine relationships between the adhesion index and titer pili 85 kDa with a limit of significance, 0.05 (p Results: The hemagglutination test showed no ability of agglutination with the formation of red aggregate points starting from the first dilution. An analysis with Pearson correlation indicated a relationship between titer pili 85 kDa E. coli and adhesion index (p = 0.009; R = -0.921).Conclusion: Pili protein 85 kDa E. coli plays a role as an adhesin protein and do not have a role as a hemagglutinin protein.