A Polyclonal Antibody against a Burkholderia cenocepacia OmpA-like Protein Strongly Impairs Pseudomonas aeruginosa and B. multivorans Virulence.

Despite advances in therapies, bacterial chronic respiratory infections persist as life-threatening to patients suffering from cystic fibrosis (CF). Pseudomonas aeruginosa and bacteria of the Burkholderia cepacia complex are among the most difficult of these infections to treat, due to factors like their resistance to multiple antibiotics and ability to form biofilms. The lack of effective antimicrobial strategies prompted our search for alternative immunotherapies that can effectively control and reduce those infections among CF patients. Previous work from our group showed that the anti-BCAL2645 goat polyclonal antibody strongly inhibited Burkholderia cenocepacia to adhere and invade cultured epithelial cells. In this work, we showed that the polyclonal antibody anti-BCAL2645 also strongly inhibited the ability of P. aeruginosa to form biofilms, and to adhere and invade the human bronchial epithelial cell line CFBE41o-. The polyclonal antibody also inhibited, to a lesser extent, the ability of B. multivorans to adhere and invade the human bronchial epithelial cell line CFBE41o. We also show that the ability of B. cenocepacia, P. aeruginosa and B. multivorans to kill larvae of the Galleria mellonella model of infection was impaired when bacteria were incubated with the anti-BCAL2645 antibody prior to the infection. Our findings show that an antibody against BCAL2645 possesses a significant potential for the development of new immunotherapies against these three important bacterial species capable of causing devastating and often lethal infections among CF patients.

A Polyclonal Antibody Raised against the Burkholderia cenocepacia OmpA-like Protein BCAL2645 Impairs the Bacterium Adhesion and Invasion of Human Epithelial Cells In Vitro.

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain a life threat to cystic fibrosis (CF) patients, due to the faster lung function decline and the absence of effective eradication strategies. Immunotherapies are regarded as an attractive alternative to control and reduce the damages caused by these infections. In this work, we report the cloning and functional characterization of the OmpA-like BCAL2645 protein, previously identified and found to be immunoreactive against sera from CF patients with a record of Bcc infections. The BCAL2645 protein is shown to play a role in biofilm formation, adherence to mucins and invasion of human lung epithelial cells. The expression of the BCAL2645 protein was found to be increased in culture medium, mimicking the lungs of CF patients and microaerophilic conditions characteristic of the CF lung. Moreover, a polyclonal antibody raised against BCAL2645 was found to inhibit, by about 75 and 85%, the ability of B. cenocepacia K56-2 to bind and invade in vitro CFBE41o- human bronchial epithelial cells. These results highlight the potential of anti-BCAL2645 antibodies for the development of passive immunization therapies to protect CF patients against Bcc infections.

A Histone-Like Nucleoid Structuring Protein Regulates Several Virulence Traits in Burkholderia multivorans.

Burkholderia cepacia complex bacteria comprise opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. These microorganisms produce an exopolysaccharide named cepacian, which is considered a virulence determinant. To find genes implicated in the regulation of cepacian biosynthesis, we characterized an evolved nonmucoid variant (17616nmv) derived from the ancestor, Burkholderia multivorans ATCC 17616, after prolonged stationary phase. Lack of cepacian biosynthesis was correlated with downregulation of the expression of bce genes implicated in its biosynthesis. Furthermore, genome sequencing of the variant identified the transposition of the mobile element IS406 upstream of the coding sequence of an hns-like gene (Bmul_0158) encoding a histone-like nucleoid structuring (H-NS) protein, a known global transcriptional repressor. This insertion sequence (IS) element upregulated the expression of Bmul_0158 by 4-fold. Transcriptome analysis identified the global effects of this mutation on gene expression, with major changes in genes implicated in motility, pilus synthesis, type VI secretion, and chromosome-associated functions. Concomitant with these differences, the nonmucoid variant displays reduced adherence to a CF lung bronchial cell line and reduced surface hydrophobicity and forms smaller cellular aggregates but has an increase in swimming and swarming motilities. Finally, analysis of the GC content of the upstream region of differentially expressed genes led to the identification of various genomic regions, possibly acquired by horizontal gene transfer, which were transcriptionally repressed by the increased expression of the Bmul_0158 gene in the 17616nmv strain. Taken together, the results revealed a significant role for this H-NS protein in the regulation of B. multivorans persistence- and virulence-associated genes. IMPORTANCE Members of the histone-like nucleoid structuring (H-NS) family of proteins, present in many bacteria, are important global regulators of gene expression. Many of the regulated genes were acquired horizontally and include pathogenicity islands and prophages, among others. Additionally, H-NS can play a structural role by bridging and compacting DNA, fulfilling a crucial role in cell physiology. Several virulence phenotypes have been frequently identified in several bacteria as dependent on H-NS activity. Here, we describe an H-NS-like protein of the opportunistic pathogen Burkholderia multivorans, a species commonly infecting the respiratory tract of cystic fibrosis patients. Our results indicate that this protein is involved in regulating virulence traits such as exopolysaccharide biosynthesis, adhesion to biotic surfaces, cellular aggregation, and motility. Furthermore, this H-NS-like protein is one out of eight orthologs present in the B. multivorans ATCC 17616 genome, posing relevant questions to be investigated on how these proteins coordinate the expression of virulence traits.

Preservation of optic nerve structure by complement inhibition in experimental glaucoma.

Glaucoma is characterized by a progressive damage of the retina and the optic nerve. Despite a huge research interest, the exact pathomechanisms are still unknown. In the experimental autoimmune glaucoma model, rats develop glaucoma-like damage of the retina and the optic nerve after immunization with an optic nerve antigen homogenate (ONA). An early activation of the complement system, even before optic nerve degeneration, was reported in this model. Here, we investigated the effects of a monoclonal antibody against complement factor C5 on optic nerves. Rats were immunized with ONA and compared to controls. In one eye of some ONA animals, the antibody against C5 was intravitreally injected (15 µmol: ONA + C5-I or 25 µmol: ONA + C5-II) before immunization and then every 2 weeks. After 6 weeks, optic nerves were processed for histology (n = 6/group). These analyses demonstrated that the intravitreal therapy reduced the depositions of the membrane attack complex compared to ONA animals (ONA + C5-I: p = 0.005; ONA + C5-II: p = 0.002). Cellular infiltration was significantly reduced in the ONA + C5-I group (p = 0.003), but not in ONA + C5-II tissues (p = 0.41). Furthermore, SMI-32 staining revealed that neurofilament was preserved in both treatment groups compared to ONA optic nerves (both p = 0.002). A decreased amount of microglia was found in treated animals in comparison to the ONA group (ONA + C5-I: p = 0.03; ONA + C5-II: p = 0.009). We observed, for the first time, that a complement system inhibition could prevent optic nerve damage in an autoimmune glaucoma model. Therefore, complement inhibition could serve as a new therapeutic tool for glaucoma.

Intravitreal Therapy Against the Complement Factor C5 Prevents Retinal Degeneration in an Experimental Autoimmune Glaucoma Model.

In glaucoma, studies revealed an involvement of the complement system. In an experimental autoimmune glaucoma model, immunization with an optic nerve homogenate antigen (ONA) led to retinal ganglion cell (RGC) loss, while intraocular pressure (IOP) remained unchanged. Here, we investigated the therapeutic effect of a complement system inhibition in this model. Hence, rats were immunized with ONA and compared to controls. In one eye of the ONA animals, an antibody against complement factor C5 was intravitreally injected (15 µmol: ONA+C5-I or 25 µmol: ONA+C5-II) before immunization and then every two weeks. IOP was measured weekly. After 6 weeks, spectral-domain optical coherence tomographies (SD-OCT), electroretinograms (ERG), immunohistochemistry, and quantitative real-time PCR analyses were performed. IOP and retinal thickness remained unchanged within all groups. The a-wave amplitudes were not altered in the ONA and ONA+C5-I groups, whereas a decrease was noted in ONA+C5-II animals (p < 0.05). ONA immunization provoked a significant decrease of the b-wave amplitude (p < 0.05), which could be preserved in ONA+C5-I, but not in ONA+C5-II animals. ONA animals showed a loss of RGCs (p = 0.001), while ONA+C5-I and ONA+C5-II retinae had similar cell counts as controls. A significant downregulation of apoptotic Bax/Bcl2 mRNA was noted in ONA+C5-I retinae (p = 0.02). Significantly more C3+ and MAC+ cells were observed in ONA animals (p < 0.001). The amount of C3+ cells in both treatment groups was significantly increased (p < 0.01), while the number of MAC+ cells in the treated retinas did not differ from controls. The number of activated microglia cells remained unchanged in ONA animals, but was increased in the treatment groups (p < 0.05). Recoverin+ cells were diminished in ONA animals (p = 0.049), but not in treated ones. Rho mRNA was downregulated in ONA and in ONA+C5-II retinas (both p = 0.014). Less opsin+ cones were observed in ONA animals (p = 0.009), but not in the treated groups. Our results indicate that the C5 antibody inhibits activation of the complement system, preventing the loss of retinal function as well as RGC, cone bipolar, and photoreceptor loss. Therefore, this approach might be a suitable new treatment for glaucoma patients, in which immune dysregulation plays an important factor for the development and progression of glaucoma.

Mucoid switch in Burkholderia cepacia complex bacteria: Triggers, molecular mechanisms and implications in pathogenesis.

Bacteria produce a vast range of exopolysaccharides (EPSs) to thrive in diverse environmental niches and often display a mucoid phenotype in solid media. One such exopolysaccharide, cepacian, is produced by bacteria of the genus Burkholderia and is of interest due to its role in pathogenesis associated with lung infections in cystic fibrosis (CF) patients. Cepacian is a repeat-unit polymer that has been implicated in biofilm formation, immune system evasion, interaction with host cells, resistance against antimicrobials, and virulence. Its biosynthesis proceeds through the Wzy-dependent polymerization and secretion mechanism, which requires a multienzymatic complex. Key aspects of its structure, genetic organization, and the regulatory network involved in mucoid switch and regulation of cepacian biosynthesis at transcriptional and posttranscriptional levels are reviewed. It is also evaluated the importance of cepacian biosynthesis/regulation key players as evolutionary targets of selection and highlighted the complexity of the regulatory network, which allows cells to coordinate the expression of metabolic functions to the ones of the cell wall, in order to be successful in ever changing environments, including in the interaction with host cells.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.