Surface Polysaccharides Research Articles

Heteroleptic pincer palladium(II) complex coated orthopedic implants impede the AbaI/AbaR quorum sensing system and biofilm development by Acinetobacter baumannii

Implant-associated infections mediated by Acinetobacter baumannii biofilms have become a major concern in the healthcare sector. As biofilm formation by this important pathogen is mediated by quorum sensing, quorum sensing inhibitors (QSI) have gained much attention. The present study confirms that novel thiazolinyl-picolinamide based palladium(II) complexes had good biofilm disruptive and QSI properties against A. baumannii. Key QS-mediated virulence factors like pili mediated surface motility and polysaccharide production were inhibited by the best Pd(II) complex (E). This also showed potent inhibitory activity against both the standard and clinical strains of A. baumannii. Molecular docking analysis also proved the potent binding affinity of Pd(II)-E with the virulence targets. The Pd(II) complex also disrupted preformed biofilms and down-regulated the expression of QS mediated virulence genes in the biofilms established on implant material (titanium plates). As a whole, the present study showed that the novel thiazolinyl-picolinamide based Pd(II) complexes offer a promising anti-infective strategy to combat biofilm-mediated implant infections.

Immunization against a Conserved Surface Polysaccharide Stimulates Bovine Antibodies with Opsonic Killing Activity but Does Not Protect against Babesia bovis Challenge.

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.

Treating Bacterial Infections with Bacteriophage-Based Enzybiotics: In Vitro, In Vivo and Clinical Application.

Over the past few decades, we have witnessed a surge around the world in the emergence of antibiotic-resistant bacteria. This global health threat arose mainly due to the overuse and misuse of antibiotics as well as a relative lack of new drug classes in development pipelines. Innovative antibacterial therapeutics and strategies are, therefore, in grave need. For the last twenty years, antimicrobial enzymes encoded by bacteriophages, viruses that can lyse and kill bacteria, have gained tremendous interest. There are two classes of these phage-derived enzymes, referred to also as enzybiotics: peptidoglycan hydrolases (lysins), which degrade the bacterial peptidoglycan layer, and polysaccharide depolymerases, which target extracellular or surface polysaccharides, i.e., bacterial capsules, slime layers, biofilm matrix, or lipopolysaccharides. Their features include distinctive modes of action, high efficiency, pathogen specificity, diversity in structure and activity, low possibility of bacterial resistance development, and no observed cross-resistance with currently used antibiotics. Additionally, and unlike antibiotics, enzybiotics can target metabolically inactive persister cells. These phage-derived enzymes have been tested in various animal models to combat both Gram-positive and Gram-negative bacteria, and in recent years peptidoglycan hydrolases have entered clinical trials. Here, we review the testing and clinical use of these enzymes.

Vaccination against the broadly expressed microbial antigen PNAG prevents cognitive decline in the APP-PS1 mouse model of Alzheimer's disease.

While there is ample evidence for a role of the microbiota in cognitive decline in Alzheimer's Disease (AD), there is little insight into how the microbiota mediates neuronal dysfunction and cognitive decline. A conserved surface polysaccharide, poly-N-acetyl glucosamine (PNAG) is produced by a broad range of microbial cells, but not mammals. We have detected the presence of PNAG-positive, likely microbiota-derived cells or fragments closely associated with amyloid-ß deposits in the brains of deceased AD individuals and cognitively impaired APP-PS1 mice, but not non-AD controls. These fragments potentially provide an inflammatory stimulus critical for neural tissue destruction and cognitive impairment. We tested whether vaccination against PNAG prevented microbial access to brains of APP-PS1 mice and impacted cognitive decline. Microbial factors were detected with a human IgG monoclonal antibody (MAb) to PNAG in brain tissues from human AD and APP/PS1 mice. Two cohorts of APP/PS1 mice and wild type (WT) littermate controls were vaccinated with either a PNAG vaccine (5GlcNH2 -TT) or TT (tetanus toxin) only as a control, five times starting at either 5 weeks or 5 months of age. Only PNAG-immunized mice made specific antibodies over the experimental period. Water T maze testing of mice was performed at 12-15 months of age. Brains were harvested to detect both PNAG and amyloid-ß immunohistochemically. PNAG-positive microbial factors were elevated in areas with amyloid-ß deposits in brain tissue from human AD cases and APP/PS1 mice. Non-AD age- and sex-matched controls had little detectable brain PNAG, even around amyloid-ß deposits. Active PNAG vaccination at both 5 weeks and 5 months of age prevented cognitive decline in APP-PS1 mice. Only 10% or 30% of non-immune APP/PS1 mice reached criteria by day 5 of reversal, respectively, compared to 90% and 100% of PNAG vaccinated APP/PS1 mice. Immunohistochemistry indicated decreased presence of PNAG-positive material in PNAG vaccinated mice and a modest reduction in amyloid-ß deposits. Antibodies to PNAG induced at 5 weeks or 5 months of age prevented cognitive decline and brain pathology in APP/PS1 mice, suggesting that microbial factors that enter the brain are critical for cognitive decline that may be prevented by vaccination.

Polysaccharide-Based Nanoparticles as Pickering Emulsifiers in Emulsion Formulations and Heterogenous Polymerization Systems.

Bio-based Pickering emulsifiers are a nontoxic alternative to surfactants in emulsion formulations and heterogenous polymerizations. Recent demand for biocompatible and sustainable formulations has accelerated academic interest in polysaccharide-based nanoparticles as Pickering emulsifiers. Despite the environmental advantages, the inherent hydrophilicity of polysaccharides and their nanoparticles limits efficiency and application range. Modification of the polysaccharide surface is often required in the development of ultrastable, functional, and water-in-oil (W/O) systems. Complex surface modification calls into question the sustainability of polysaccharide-based nanoparticles and is identified as a significant barrier to commercialization. This review summarizes the use of nanocelluloses, -starches, and -chitins as Pickering emulsifiers, highlights trends and best practices in surface modification, and provides recommendations to expedite commercialization.

Development of a Molecular Serotyping Scheme for Morganella morganii.

Morganella morganii, which is often regarded as a human commensal organism, can be an opportunistic pathogen, causing a variety of clinical infections with serious morbidity and mortality. An efficient and convenient method for subtyping and identifying M. morganii strains in epidemiological surveillance and control is urgently needed. Serotyping based on bacterial surface polysaccharide antigens (O-antigen or K-antigens) is a standard subtyping method for many gram-negative bacteria. Here, through whole genome sequencing and comparative genomics analysis of 27 strains, we developed a molecular serotyping scheme based on the genetic variation of O-antigen gene clusters (O-AGC) in M. morganii, and 11 distinct O-AGC types were identified. A conventional serotyping scheme was also developed by the production of antisera and agglutination experiments, which was shown to be perfectly consistent with the molecular serotyping scheme, confirming that the variation in M. morganii O-AGC correlated with phenotypic O-antigen diversification. Furthermore, a microsphere-based suspension array (MSA) with high specificity was developed based on the specific genes within each O-AGC type. The sensitivity of MSA was determined to be 0.1 ng of genomic DNA and 103 CFU of pure culture. We further analyzed 104 M. morganii genomes available in GenBank, and an additional six novel O-AGC types were identified, indicating that the extension of this molecular serotyping scheme is convenient. Our work provides an important tool for the detection and epidemiological surveillance of M. morganii, and this method has the potential to be widely utilized, especially for bacterial genera/species without an efficient typing approach.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Effect of mono- and double-layer polysaccharide surface coating on the physical stability of nanoliposomes under various environments

Physical stability is an important parameter concerning the applications of nanoliposomes (NLs) in manufacturing, such as the food, cosmetic and pharmaceutical industries, since the viability of the usage of NLs to protect the activity of targeted molecules and control their release depends on it. In this work, the physical stability of reduced glutathione (GSH) loaded NLs (Lip), mono-layer chitosan coated Lip (CTS-Lip) and double-layer alginate-chitosan coated Lip (ALG-CTS-Lip) in different conditions was characterized by the changes of the average particle sizes, zeta potentials, and leakages of the encapsulated GSH. It was revealed that pH had a negligible influence on Lip but induced significant changes in the mean particle sizes of CTS-Lip and ALG-CTS-Lip, which was inferred to relate to the solubility of ALG and CTS at different pH values. In the case of metal ions, Mg 2+ showed some influence on Lip and CTS-Lip while Zn 2+ and Ca 2+ usually used as crosslinker exhibited significant impact on CTS-Lip and ALG-CTS-Lip. According to the release rates of GSH and the changes of particle size, ALG-CTS-Lip were the most stable sample over a wide range of temperature (20–100 °C) and a long time of storage (60 days). Moreover, CTS monolayer and ALG-CTS double layer coating on the surface of NLs significantly enhanced the thermal stability of NLs and reduced its disintegration in simulated intestinal fluid (SIF). Therefore, surface coating could induce different effects on the physical stability of NLs under different environments. These results may to some extent facilitate the rational design of novel NL-based delivery systems used in manufacturing. • Different environmental stressors like pH, temperature, ions and enzymes were studied. • The physical stability of Lip, CTS-Lip and ALG-CTS-Lip changed in different conditions. • Mono- and double-layer polysaccharide surface coating exhibited different effects.

Structural properties and hydrolysability of recycled poplar residues (Populus L.): Effects of two-step acetic acid and sodium sulphite pre-treatment

Poplar trees rapidly yield wood and are therefore suitable as a biofuel feedstock; however, the quality of poplar is modest, and the profitability of poplar cultivation depends on the efficiency of the harvesting process. This study offers a simple and sustainable technique to harvest lignocellulosic resources from poplar for bioethanol production. The proposed two-step pretreatment method increased the surface lignin content and decreased the surface polysaccharide content. The cellulose content increased to 54.9% and the xylan content decreased to 6.7% at 5% AC. The cellulose yield of poplar residues (Populus L.) reached 65.5% by this two-step acetic acid (AC) and sodium sulphite (SS) treatment method. Two-step pretreatment using 5% AC and 4% SS obtained a recovery of nearly 80% of the total available fermentable sugar. The surface characterization showed a higher porosity in treated samples, which improved their hydrolysability. This method decreased the amount of lignin in plant biomass, making it applicable for further wood resource recovery or waste recycling for biorefinery purposes at very low costs.

The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae.

Klebsiella pneumoniae is considered one of the most critical multidrug-resistant pathogens and urgently requires new therapeutic strategies. Capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) are the major virulence factors protecting K. pneumoniae against the immune response and thus may be targeted by phage-based therapeutics such as polysaccharides-degrading enzymes. Since the emergence of resistance to antibacterials is generally considered undesirable, in this study, the genetic and phenotypic characteristics of resistance to the phage-borne CPS-degrading depolymerase and its effect on K. pneumoniae virulence were investigated. The K63 serotype targeting depolymerase (KP36gp50) derived from Klebsiella siphovirus KP36 was used as the selective agent during the treatment of K. pneumoniae 486 biofilm. Genome-driven examination combined with the surface polysaccharide structural analysis of resistant mutant showed the point mutation and frameshift in the wbaP gene located within the cps gene cluster, resulting in the loss of the capsule. The sharp decline in the yield of CPS was accompanied by the production of a larger amount of smooth LPS. The modification of the surface polysaccharide layers did not affect bacterial fitness nor the insensitivity to serum complement; however, it made bacteria more prone to phagocytosis combined with the higher adherence and internalization to human lung epithelial cells. In that context, it was showed that the emerging resistance to the antivirulence agent (phage-borne capsule depolymerase) results in beneficial consequences, i.e., the sensitization to the innate immune response.

The S Protein of Group B Streptococcus Is a Critical Virulence Determinant That Impacts the Cell Surface Virulome.

Group B Streptococcus (GBS, S. agalactiae) is a human commensal and occasional pathogen that remains a leading cause of neonatal sepsis and meningitis with increasing disease burden in adult populations. Although programs for universal screening in pregnancy to guide intrapartum prophylaxis have reduced GBS invasive disease burden resulting from mother-to-newborn transfer during birth, better knowledge of disease mechanisms may elucidate new strategies to reduce antibiotic exposure. In our efforts to expand the knowledge base required for targeted anti-virulence therapies, we identified a GBS homolog for a recently identified virulence determinant of group A Streptococcus, S protein, and evaluated its role in GBS pathogenesis. A GBS S protein deletion mutant, Δess, showed altered cell-surface properties compared to the WT parent strain, including defective retention of its surface polysaccharide. Quantitative proteome analysis of enzymatically shaved surface epitopes of the GBS Δess mutant revealed a dysregulated cell surface virulome, with reduced abundance of several protein and glycoprotein components. The Δess mutant showed markedly attenuated virulence in a murine model of GBS systemic infection, with increased proteasome activity detected in the spleens of animals infected with the Δess mutant. These results expand the key roles S protein plays in streptococcal pathogenesis and introduces a new GBS virulence determinant and potential target for therapy development.

Cell Surface Polysaccharides Represent a Common Strategy for Adsorption among Phages Infecting Lactic Acid Bacteria: Lessons from Dairy Lactococci and Streptococci.

ABSTRACTFood fermentations rely on the application of robust bacterial starter cultures, the majority of which are represented by members of the lactic acid bacteria including Lactococcus lactis and Streptococcus thermophilus. Bacteriophage (or phage) proliferation remains one of the most significant threats to the fermentation industry. Therefore, it is imperative to define the phage ecology of fermented foods and to elucidate the mechanisms by which they recognize and bind to their bacterial hosts. Through a combination of functional and comparative genomics and structural analysis of the phage-host interactome, it is now possible to link the genotypes of strains of certain bacterial species to the chemical composition/structure of the associated cell wall polysaccharides (CWPS). In this paper, I discuss how the identification of common host recognition and binding strategies facilitates the development of rational starter culture systems and the implications of these findings in the context of sustainable food production systems.

Antibody activities in hyperimmune plasma against the Rhodococcus equi virulence -associated protein A or poly-N-acetyl glucosamine are associated with protection of foals against rhodococcal pneumonia.

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide β-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

Structural characterization of a novel pentasaccharide repeating unit from Burkholderia pseudomallei strain BPC006 and its role in diagnosis and immunogenicity

Burkholderia pseudomallei causes melioidosis - an infectious disease with high mortality. Its varied clinical manifestations and resistance to many antibiotics make it a potential biothreat agent and calls for a robust diagnostic assay and effective vaccines. Bacterial cell surface polysaccharides are considered a valuable target for diagnostics and as protective antigen candidates. This study characterized the structure of polysaccharides of B. pseudomallei clinical strain from Hainan, China. A novel structural domain [→3-(α-D-Manp-1→3-α-D-Manp)2-2Me-α-L-6dTalp-1→] was identified by chemical analysis, gas chromatography-mass spectrometry (GC-MS), and 1D/2D nuclear magnetic resonance (NMR) spectroscopy. Immunofluorescence and enzyme-linked immunosorbent assay (ELISA) showed that the serum antibodies against the purified polysaccharide antigen could recognize and bind specifically to B. pseudomallei strains. Additionally, the assays revealed cross-reactivity with polysaccharides from different clinical strains. The polysaccharide antigen also exhibited a strong reaction with the sera from melioidosis patients. Thus, the pentasaccharide repeating unit residue could be a potential candidate antigen for the melioidosis serodiagnosis and vaccine development.

Genomic and molecular evidence reveals novel pathways associated with cell surface polysaccharides in bacteria.

Amino acid (acyl carrier protein) ligases (AALs) are a relatively new family of bacterial amino acid adenylating enzymes with unknown function(s). Here, genomic enzymology tools that employ sequence similarity networks and genome context analyses were used to hypothesize the metabolic function(s) of AALs. In over 50% of species, aal and its cognate acyl carrier protein (acp) genes, along with three more genes, formed a highly conserved AAL cassette. AAL cassettes were strongly associated with surface polysaccharide gene clusters in Proteobacteria and Actinobacteria, yet were prevalent among soil and rhizosphere-associated α- and β-Proteobacteria, including symbiotic α- and β-rhizobia and some Mycolata. Based on these associations, AAL cassettes were proposed to encode a noncanonical Acp-dependent polysaccharide modification route. Genomic-inferred predictions were substantiated by published experimental evidence, revealing a role for AAL cassettes in biosynthesis of biofilm-forming exopolysaccharide in pathogenic Burkholderia and expression of aal and acp genes in nitrogen-fixing Rhizobium bacteroids. Aal and acp genes were associated with dltBD-like homologs that modify cell wall teichoic acids with d-alanine, including in Paenibacillus and certain other bacteria. Characterization of pathways that involve AAL and Acp may lead to developing new plant and human disease-controlling agents as well as strains with improved nitrogen fixation capacity.

Serum Antibody Activity against Poly-N-Acetyl Glucosamine (PNAG), but Not PNAG Vaccination Status, Is Associated with Protecting Newborn Foals against Intrabronchial Infection with Rhodococcus equi.

ABSTRACTRhodococcus equi is a prevalent cause of pneumonia in foals worldwide. Our laboratory has demonstrated that vaccination against the surface polysaccharide β-1→6-poly-N-acetylglucosamine (PNAG) protects foals against intrabronchial infection with R. equi when challenged at age 28 days. However, it is important that the efficacy of this vaccine be evaluated in foals when they are infected at an earlier age, because foals are naturally exposed to virulent R. equi in their environment from birth and because susceptibility is inversely related to age in foals. Using a randomized, blind experimental design, we evaluated whether maternal vaccination against PNAG protected foals against intrabronchial infection with R. equi 6 days after birth. Vaccination of mares per se did not significantly reduce the incidence of pneumonia in foals; however, activities of antibody against PNAG or for deposition of complement component 1q onto PNAG was significantly (P < 0.05) higher among foals that did not develop pneumonia than among foals that developed pneumonia. Results differed between years, with evidence of protection during 2018 but not 2020. In the absence of a licensed vaccine, further evaluation of the PNAG vaccine is warranted, including efforts to optimize the formulation and dose of this vaccine.IMPORTANCE Pneumonia caused by R. equi is an important cause of disease and death in foals worldwide for which a licensed vaccine is lacking. Foals are exposed to R. equi in their environment from birth, and they appear to be infected soon after parturition at an age when innate and adaptive immune responses are diminished. Results of this study indicate that higher activity of antibodies recognizing PNAG was associated with protection against R. equi pneumonia, indicating the need for further optimization of maternal vaccination against PNAG to protect foals against R. equi pneumonia.

Evaluation of cell wall-associated direct extracellular electron transfer in thermophilic Geobacillus sp.

The online version contains supplementary material available at 10.1007/s13205-021-02917-2.

Single-Point Mutations in Qβ Virus-like Particles Change Binding to Cells.

Virus-like particles (VLPs) constitute large, polyvalent platforms onto which a wide variety of functional units can be grafted. Their use in biological settings often depends on their specific binding to cells or receptors of interest; this can be compromised by excessive nonspecific association with other cells. We found that lysine residues mediate such nonspecific interactions, presumably by virtue of protonation and interaction with anionic membrane lipid headgroups and/or complementary residues of cell surface proteins and polysaccharides. Chemical acylation of surface-exposed amines of the Qβ VLP led to a significant reduction in the association of particles with mammalian cells. Single-point mutations of particular lysine residues to either glutamine, glutamic acid, tryptophan, or phenylalanine were mostly well-tolerated and formed intact capsids, but the introduction of double and triple mutants was far less forgiving. Introduction of glutamic acid at position 13 (K13E) led to a dramatic increase in cellular binding, whereas removal of the lysine at position 46 (K46Q) led to an equally striking reduction. Several plasma membrane components were found to specifically interact with the Qβ capsid irrespective of surface charge. These results suggest that specific cellular interactions are engaged or obviated by such mutations and provide us with more "benign" particles to which can be added binding functionality for targeted delivery applications.

Pectobacterium parmentieri SCC 3193 Mutants with Altered Synthesis of Cell Surface Polysaccharides Are Resistant to N4-Like Lytic Bacteriophage ϕA38 (vB_Ppp_A38) but Express Decreased Virulence in Potato (Solanum tuberosum L.) Plants.

Pectobacterium parmentieri is a Gram-negative plant-pathogenic bacterium able to infect potato (Solanum tuberosum L.). Little is known about lytic bacteriophages infecting P. parmentieri and how phage-resistance influences the environmental fitness and virulence of this species. A lytic phage vB_Ppp_A38 (ϕA38) has been previously isolated and characterized as a potential biological control agent for the management of P. parmentieri. In this study, seven P. parmentieri SCC 3193 Tn5 mutants were identified that exhibited resistance to infection caused by vB_Ppp_A38 (ϕA38). The genes disrupted in these seven mutants encoded proteins involved in the assembly of O-antigen, sugar metabolism, and the production of bacterial capsule exopolysaccharides. The potential of A38-resistant P. parmentieri mutants for plant colonization and pathogenicity as well as other phenotypes expected to contribute to the ecological fitness of P. parmentieri, including growth rate, use of carbon and nitrogen sources, production of pectinolytic enzymes, proteases, cellulases, and siderophores, swimming and swarming motility, presence of capsule and flagella as well as the ability to form biofilm were assessed. Compared to the wild-type P. parmentieri strain, all phage-resistant mutants exhibited a reduced ability to colonize and to cause symptoms in growing potato (S. tuberosum L.) plants. The implications of bacteriophage resistance on the ecological fitness of P. parmentieri are discussed.

Immunoglobulin GM and KM allotypes are associated with antibody responses to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients

Chronic infection with Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of death in patients with cystic fibrosis (CF). Immunobiology of P. aeruginosa infection is complex and not well understood. Chronically infected CF patients generate high levels of antibodies to P. aeruginosa, but this response does not lead to clinical improvement. Therefore, additional studies aimed at identification and understanding of the host factors that influence naturally occurring immune responses to P. aeruginosa are needed. In this investigation, we evaluated the contribution of immunoglobulin GM (γ marker) and KM (κ marker) allotypes to the antibody responses to P. aeruginosa lipopolysaccharide (LPS) O1, O6, O11, and alginate antigens and the broadly-conserved surface polysaccharide expressed by many microbial pathogens, poly-N-acetyl-D-glucosamine (PNAG), in 58 chronically infected CF patients. IgG1 markers GM 3 and 17 and IgG2 markers GM 23- and 23+ were determined by a pre-designed TaqMan® genotyping assay. The κ chain determinants KM 1 and 3 were characterized by PCR-RFLP. Antibodies to the LPS O antigens, alginate, and PNAG were measured by an ELISA. Several significant associations were noted with KM alleles. Particular KM 1/3 genotypes were individually and epistatically (with GM 3/17) associated with the level of IgG antibodies to O1, O11, alginate, and PNAG antigens. Immunoglobulin GM and KM genotypes influence the magnitude of humoral immunity to LPS O, alginate, and PNAG antigens. These results, if confirmed in a larger study population, will be helpful in devising novel immunotherapeutic approaches against P. aeruginosa.