Browsing by Author "Inzana, Thomas J."
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- Antibiofilm Activity of Actinobacillus pleuropneumoniae Serotype 5 Capsular PolysaccharideKarwacki, Michael T.; Kadouri, Daniel E.; Bendaoud, Meriem; Izano, Era A.; Sampathkumar, Vandana; Inzana, Thomas J.; Kaplan, Jeffrey B. (PLOS, 2013-05-14)Cell-free extracts isolated from colony biofilms of Actinobacillus pleuropneumoniae serotype 5 were found to inhibit biofilm formation by Staphylococcus aureus, S. epidermidis and Aggregatibacter actinomycetemcomitans, but not by A. pleuropneumoniae serotype 5 itself, in a 96-well microtiter plate assay. Physical and chemical analyses indicated that the antibiofilm activity in the extract was due to high-molecular-weight polysaccharide. Extracts isolated from a mutant strain deficient in the production of serotype 5 capsular polysaccharide did not exhibit antibiofilm activity. A plasmid harboring the serotype 5 capsule genes restored the antibiofilm activity in the mutant extract. Purified serotype 5 capsular polysaccharide also exhibited antibiofilm activity against S. aureus. A. pleuropneumoniae wild-type extracts did not inhibit S. aureus growth, but did inhibit S. aureus intercellular adhesion and binding of S. aureus cells to stainless steel surfaces. Furthermore, polystyrene surfaces coated with A. pleuropneumoniae wild-type extracts, but not with capsule-mutant extracts, resisted S. aureus biofilm formation. Our findings suggest that the A. pleuropneumoniae serotype 5 capsule inhibits cell-to-cell and cell-to-surface interactions of other bacteria. A. pleuropneumoniae serotype 5 capsular polysaccharide is one of a growing number of bacterial polysaccharides that exhibit broad-spectrum, nonbiocidal antibiofilm activity. Future studies on these antibiofilm polysaccharides may uncover novel functions for bacterial polysaccharides in nature, and may lead to the development of new classes of antibiofilm agents for industrial and clinical applications.
- Antigenic Characterization of Haemophilus somnus LipooligosaccharideHoward, Michael D. (Virginia Tech, 1998-10-23)Lipooligosaccharide (LOS) is the major outer membrane component of many Gram-negative bacteria inhabiting the mucosal membranes, including pathogenic species of Haemophilus and Neisseria. LOS phase variation is one mechanism by which some of these bacteria avoid the host immune response. To better understand LOS phase variation as a virulence mechanism of H. somnus, knowledge of the antigenic diversity of LOS epitopes must be increased. Monoclonal antibodies (MAbs) to H. somnus LOS were produced and used with cross-reacting MAbs to H. aegyptius LOS (MAb 5F5) and Neisseria gonorrhoeae LOS (MAb 3F11) in an ELISA to investigate LOS heterogeneity among forty-five strains of H. somnus. Using three MAbs, thirty-nine of these H. somnus strains were grouped into six antigenic types. Three groups, associated solely with the cross-reacting MAbs 5F5 and 3F11, included the majority (76%) of H. somnus strains. The anti-H. somnus LOS MAb 5D7 recognized a low frequency epitope associated with each of the remaining three groups, which included 11% of the H. somnus strains. Six strains (13%) were not recognized by any of these MAbs. Inhibition ELISA experiments showed that the MAb 5F5 epitope contained phosphocholine (PCho) and this epitope was present in 56% of the strains tested. The MAb 5F5 epitope is phase variable in H. somnus LOS. How PCho negative variants could allow for systemic infection after initial colonization of the mucosa by PCho positive variants is discussed.
- The Application of a Nanomaterial Optical Fiber Biosensor Assay for Identification of Brucella NomenspeciesMcCutcheon, Kelly; Bandara, Aloka B.; Zuo, Ziwei; Heflin, James R.; Inzana, Thomas J. (MDPI, 2019-05-21)Bacteria in the genus Brucella are the cause of brucellosis in humans and many domestic and wild animals. A rapid and culture-free detection assay to detect Brucella in clinical samples would be highly valuable. Nanomaterial optical fiber biosensors (NOFS) are capable of recognizing DNA hybridization events or other analyte interactions with high specificity and sensitivity. Therefore, a NOFS assay was developed to detect Brucella DNA from cultures and in tissue samples from infected mice. An ionic self-assembled multilayer (ISAM) film was coupled to a long-period grating optical fiber, and a nucleotide probe complementary to the Brucella IS711 region and modified with biotin was bound to the ISAM by covalent conjugation. When the ISAM/probe duplex was exposed to lysate containing ≥100 killed cells of Brucella, or liver or spleen tissue extracts from Brucella-infected mice, substantial attenuation of light transmission occurred, whereas exposure of the complexed fiber to non-Brucella gram-negative bacteria or control tissue samples resulted in negligible attenuation of light transmission. Oligonucleotide probes specific for B. abortus, B. melitensis, and B. suis could also be used to detect and differentiate these three nomenspecies. In summary, the NOFS biosensor assay detected three nomenspecies of Brucella without the use of polymerase chain reaction within 30 min and could specifically detect low numbers of this bacterium in clinical samples.
- Capsular Polysaccharide Interferes with Biofilm Formation by Pasteurella multocida Serogroup APetruzzi, Briana; Briggs, Robert E.; Swords, W. Edward; De Castro, Cristina; Molinaro, Antonio; Inzana, Thomas J. (American Society for Microbiology, 2017-11)Pasteurella multocida is an important multihost animal and zoonotic pathogen that is capable of causing respiratory and multisystemic diseases, bacteremia, and bite wound infections. The glycosaminoglycan capsule of P. multocida is an essential virulence factor that protects the bacterium from host defenses. However, chronic infections (such as swine atrophic rhinitis and the carrier state in birds and other animals) may be associated with biofilm formation, which has not been characterized in P. multocida. Biofilm formation by clinical isolates was inversely related to capsule production and was confirmed with capsule-deficient mutants of highly encapsulated strains. Capsule-deficient mutants formed biofilms with a larger biomass that was thicker and smoother than the biofilm of encapsulated strains. Passage of a highly encapsulated, poor-biofilm-forming strain under conditions that favored biofilm formation resulted in the production of less capsular polysaccharide and a more robust biofilm, as did addition of hyaluronidase to the growth medium of all of the strains tested. The matrix material of the biofilm was composed predominately of a glycogen exopolysaccharide (EPS), as determined by gas chromatography-mass spectrometry, nuclear magnetic resonance, and enzymatic digestion. However, a putative glycogen synthesis locus was not differentially regulated when the bacteria were grown as a biofilm or planktonically, as determined by quantitative reverse transcriptase PCR. Therefore, the negatively charged capsule may interfere with biofilm formation by blocking adherence to a surface or by preventing the EPS matrix from encasing large numbers of bacterial cells. This is the first detailed description of biofilm formation and a glycogen EPS by P. multocida. IMPORTANCE Pasteurella multocida is an important pathogen responsible for severe infections in food animals, domestic and wild birds, pet animals, and humans. P. multocida was first isolated by Louis Pasteur in 1880 and has been studied for over 130 years. However, aspects of its lifecycle have remained unknown. Although formation of a biofilm by P. multocida has been proposed, this report is the first to characterize biofilm formation by P. multocida. Of particular interest is that the biofilm matrix material contained a newly reported amylose-like glycogen as the exopolysaccharide component and that production of capsular polysaccharide (CPS) was inversely related to biofilm formation. However, even highly mucoid, poor-biofilm-forming strains could form abundant biofilms by loss of CPS or following in vitro passage under biofilm growth conditions. Therefore, the carrier state or subclinical chronic infections with P. multocida may result from CPS downregulation with concomitant enhanced biofilm formation.
- The capsular polysaccharide of Actinobacillus pleuropneumoniae serotype 5A: role in serum resistance and characterization of the genetic basis for expressionWard, Christine K. (Virginia Tech, 1995)Actinobacillus pleuropneumoniae synthesizes a serotype-specific capsular polysaccharide (CP) that protects this bacterium from host defenses. In the presence of anti-CP IgG, encapsulated A. pleuropneumoniae K17 was killed in precolostral calf serum (PCS) but not in normal serum used as a complement source. In contrast, two capsule-deficient mutants were killed in normal serum. The CP of A. pleuropneumoniae contributed to serum-resistance by limiting the amount of C9, a component of the membrane attack complex, but not C3, that bound to the bacteria in PCS. A second mechanism of serum resistance was due to a lipopolysaccharide (LPS)-specific antibody present in the IgG fractions of normal swine serum, swine anti-K17 serum, and guinea pig anti-K17 LPS serum that blocked anti-CP IgG complement-mediated killing of A. pleuropneumoniae. This LPS-specific antibody prevented complement-mediated killing of K17 in the presence of potentially bactericidal anti-CP IgG by reducing the deposition of C9 onto A. pleuropneumoniae, and by directing the deposition of C9 to sites on the bacteria where the bound C9 was easily eluted. Thus, CP and anti-LPS antibody may act synergistically or at different stages of infection to limit the ability of complement to eliminate A. pleuropneumoniae. Two overlapping regions of the A. pleuropneumoniae J45 capsulation locus were cloned and partially sequenced. One region was conserved among A. pleuropneumoniae serotypes and contained four open reading frames, cpxDCBA, that were highly homologous at both the nucleotide and amino acid levels to genes involved in the export of the CP of H. influenzae type b (bexDCBA), Neisseria meningitidis group B (ctrABCD), and to a lesser extent Escherichia coli K1 and K5 (kpsED, kpsMT). The J45 cpxDCBA gene cluster was able to partially complement kpsM::TnphoA or kpsT::TnphoA mutations within a plasmid-encoded E. coli K5 kps locus and restored sensitivity to a K5-specific bacteriophage, indicating that cpxDCBA functioned in capsular polysaccharide export. A DNA region adjacent to A. pleuropneumoniae J45 cpxDCBA was identified that was serotype-specific. This region contained two complete open reading frames (cpsA and cpsB), and a third partial open reading frame, cpsC. These genes may encode proteins involved in A. pleuropneumoniae J45 CP biosynthesis. A recombinant A. pleuropneumoniae J45 mutant in which the three serotype-specific genes, cpsABC, were partially or completely deleted was generated by allelic exchange. This mutant did not produce intracellular or extracellular CP, was serum-sensitive, and was attenuated in pigs. These studies demonstrated that CP contributed to the serum-resistance and virulence of A. pleuropneumoniae. This noncapsulated mutant will be evaluated as a potential live vaccine strain for the control of swine pleuropneumonia.
- Characterization of Deoxycholate-Responsive Genes Utilized by Brucella abortus 2308 During Oral InfectionLehman, Christian Ryan (Virginia Tech, 2017-07-17)Brucellosis is a chronic, recurring disease caused by the bacterium Brucella abortus, along with other species of the genus Brucella, and is one of the most common bacterial zoonosis worldwide. The bacteria preferentially infect and reside within host macrophages, causing an undulant fever, joint pain, and other flu-like symptoms, in addition to more severe problems like hepatosplenomegaly and endocarditis. Brucella infection is most often acquired via inhalation through the respiratory route, or via consumption of unpasteurized dairy products. Although ingestion is a major route of infection, the transcriptional response of B. abortus during oral infection remains poorly characterized. In this project, RNA sequencing was used to discover genes with the greatest transcriptional changes in B. abortus subjected to deoxycholate, a host bile acid encountered by bacteria during oral infection. Gene deletion strains of B. abortus were then created and tested for susceptibility to pH and bile acid stress, along with their ability to invade and replicate within macrophages. If the genes of interest are important for the oral infection process, B. abortus strains lacking these genes will likely be more susceptible to pH and deoxycholate stress and may exhibit attenuation in the macrophage infection model. Determination of genes important for the oral infection process would further elucidate the molecular mechanisms by which B. abortus invades the host, and could help lead to future treatments and novel therapeutics.
- Characterization of the Capsular Polysaccharide of Haemophilus parasuis and its Application in the Diagnosis and Prevention of Glasser's DiseaseHyman, Anne Catherine Michalenka (Virginia Tech, 2015-04-20)Haemophilus parasuis is a Gram-negative bacterium responsible for Glasser's Disease in pigs, though little is known regarding its antigenic or virulence factors. Our goals were to characterize the H. parasuis capsular polysaccharide (CP), determine its role in serotype-specificity and virulence, determine if CP is immunogenic, and develop diagnostic and protective products to prevent rampant H. parasuis infection within swine herds. Material from H. parasuis was purified using carbohydrate isolation techniques and compared to CPs from other Pasteurellaceae. Rabbits were immunized with CPs to generate antisera for microscopy, immunoassays, and bactericidal assays. CP antisera were conjugated to latex particles to create an agglutination assay for detection and typing of H. parasuis. CP was conjugated to Cholera Toxin B, and used to immunize mice and piglets before challenge with H. parasuis to determine its protective efficacy against Glasser's Disease. Broth-grown cells expressed CP, which reacted with antisera in microscopy and immunoassays. Broth-grown H. parasuis cells were serum-resistant unless homologous anti-CP serum was present. In contrast, agar-grown cells did not react with antisera in immunoassays, and cells were susceptible to killing by normal swine serum. CP was not expressed on the surface of agar-grown cells unless supplemented with bicarbonate. The addition of bicarbonate also contributed to the variability in CP quantity and upregulation of genes in the CP locus. Sensitized latex particles agglutinated strongest with homologous H. parasuis CPs, cells, and agar-grown cell lysates, but also reacted weakly with higher concentrations of heterologous CPs. The latex beads did not agglutinate with non-H. parasuis swine bacterial pathogens. Mice immunized with the CP-CTB conjugate produced a significantly higher IgG2/Th2 response than unimmunized mice or mice immunized with only CP, and immunized mice had fewer bacteria in their tissues that unimmunized mice. The CP conjugate produced a robust IgG antibody response to CP when used to immunized piglets, but because the control animals also survived H. parasuis challenge, the protective efficacy remains inconclusive. Therefore, the H. parasuis CP is the antigen that confers serotype identity, and can be implemented in methods and help direct future research in disease prevention and serotype tracking in H. parasuis infections.
- Cloning and Characterization of a Gene Involved in Lipooligosaccharide Biosynthesis in Haemophilus somnusHensley, Jennifer A. (Virginia Tech, 1998-05-06)Repetitive tetramers of the DNA sequence 5'-CAAT-3' are present in several loci associated with lipooligosaccharide (LOS) phase variation in Haemophilus influenzae type b (Hib). In an attempt to identify H. somnus phase-variable LOS genes, the presence of CAAT repeats within the H. somnus 738 genome was confirmed using a (CAAT)7 probe. A 3.9 kb EcoRI fragment that reacted with the probe was cloned and sequenced. Sequence analysis confirmed the presence of 31 CAAT repeats downstream of two potential start codons, and indicated that small or large proteins would be encoded depending on the number of CAAT repeats. The larger gene products showed 46% amino acid homology to Lex2b from Hib, which influences LOS phase variation in that species. In H. somnus, this gene was named lob1 (lipooligosaccharide biosynthesis gene). Sequence analysis showed that randomly selected colonies most frequently contained 33 CAAT repeats in lob1, corresponding to a 294 amino acid product. Colonies selected for negative reactivity to mAb 5F5 were significantly more likely to have different numbers of CAAT repeats in lob1 than randomly selected colonies. The presence of lob1 in trans altered the LOS profile of a non-phase variable strain of H. somnus, and caused increased levels of reactivity to polyclonal antisera made to purified LOS from strain 738. Based on the ability of this gene to alter the LOS profile of a non-phase varying strain and the correlation of changes in CAAT repeats with mAb 5F5 reactivity, lob1 appears to be involved in LOS biosynthesis and phase variation.
- Comparative and Functional Genomic Studies of Histophilus somni (Haemophilus somnus)Siddaramappa, Shivakumara Swamy (Virginia Tech, 2007-04-09)Histophilus somni is a commensal of the mucosal surfaces of respiratory and reproductive tracts of cattle and sheep. However, as an opportunistic pathogen, H. somni can cause diseases such as pneumonia, myocarditis, abortion, arthritis, and meningo-encephalitis. Previously, several virulence factors/mechanisms had been identified in H. somni of which the phase-variable lipooligosaccharide, induction of host cell apoptosis, intraphagocytic survival, and immunoglobulin Fc binding proteins were well characterized. To further understand the biological properties of H. somni, the genomes of pneumonia strain 2336 and preputial strain 129Pt have been sequenced. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, putative genes that encode proteases, restriction-modification enzymes, hemagglutinins, glycosyltransferases, kinases, helicases, and adhesins have been identified in H. somni. Most of the H. somni strain-specific genes were found to be associated with prophage-like sequences, plasmids, and/or transposons. Therefore, it is likely that these mobile genetic elements played a significant role in creating genomic diversity and phenotypic variability among strains of H. somni. Functional characterization of H. somni luxS in the genomic context revealed that the gene encodes S-ribosylhomocysteinase that can complement biosynthesis of AI-2 quorum sensing signal molecules in Escherichia coli DH5alpha. It was also found that several pathogenic isolates of H. somni form a prominent biofilm and that luxS as well as phosphorylcholine expression can influence biofilm formation by H. somni. In conclusion, comparative analyses of the genomes and functional characterization of putative genes have shed new light on the versatility and evolution of H. somni.
- Comparative Genomics of Early-Diverging Brucella Strains Reveals a Novel Lipopolysaccharide Biosynthesis PathwayWattam, Alice R.; Inzana, Thomas J.; Williams, Kelly P.; Mane, Shrinivasrao P.; Shukla, Maulik; Almeida, Nalvo F.; Dickerman, Allan W.; Mason, Steven; Moriyon, Ignacio; O'Callaghan, David; Whatmore, Adrian M.; Sobral, Bruno; Tiller, Rebekah V.; Hoffmaster, Alex R.; Frace, Michael A.; De Castro, Cristina; Molinaro, Antonio; Boyle, Stephen M.; De, Barun K.; Setubal, Joao C. (American Society for Microbiology, 2012-11)Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. IMPORTANCE This report examines differences between genomes from four new Brucella strains and those from the classic Brucella spp. Our results show that the four new strains are outliers with respect to the previously known Brucella strains and yet are part of the genus, forming two new clades. The analysis revealed important information about the evolution and survival mechanisms of Brucella species, helping reshape our knowledge of this important zoonotic pathogen. One discovery of special importance is that one of the strains, BO2, produces an O-antigen distinct from any that has been seen in any other Brucella isolates to date.
- Detection and Identification of Acinobacillus pleuropneumoniae serotype 5 by multiplex Polymerase Chain ReactionLo, Terry (Virginia Tech, 1997-07-31)Traditional serologic assays of Actinobacillus pleuropeumoniae often have problems with cross-reactivity. To avoid the complications of antibody-antigen reactions, a PCR assay was developed to detect Actinobacillus pleuropneumoniae and identify serotype 5 strains. Primers specific to the conserved capsular export region of A. pleuropneumoniae amplified a 0.7 kb DNA band in all strains with the exception of serotype 4. A second set of primers specific to the unique capsular biosynthesis region of serotype 5 amplified a unique 1.1 kb band for serotype 5 only. The sensitivity of this assay was determined to be less than 100 colony forming units. This PCR assay enables us to detect A. pleuronpeumoniae and definitively distinguishes serotype 5 strains from other serotyes.
- Detection of Actinobacillus Pleuropneumoniae and Identification of Serotypes 1, 2, and 8 by Multiplex Polymerase Chain ReactionSchuchert, Jennifer Ann (Virginia Tech, 2002-06-26)Traditional immunological assays used to serotype Actinobacillus pleuropneumoniae have been problematic due to cross- reactivity between serotypes, particularly serotypes 6 and 8. To avoid these serological cross-reactions, a multiplex PCR assay was developed to detect A. pleuropneumoniae and identify serotypes 1, 2, and 8. Primers specific to the conserved capsular polysaccharide export region of A. pleuropneumoniae serotype 5 amplified a 880 bp fragment in all serotypes excluding serotype 4 or a 489 bp DNA fragment in all serotypes including serotype 4. Primers specific to the capsular polysaccharide biosynthesis regions of A. pleuropneumoniae serotypes 1, 2, and 8 amplified a 1.6 kb, a 1.7 kb, and 970 bp fragment in the respective serotype. This PCR assay detects A. pleuropneumoniae and identifies serotypes 1, 2, and 8.
- Development of an Optical Fiber Biosensor with Nanoscale Self-Assembled Affinity LayerZuo, Ziwei (Virginia Tech, 2014-01-29)Optical sensor systems that integrate Long-Period-Gratings (LPG) as the detection arm have been proven to be highly sensitive and reliable in many applications. With increasing public recognition of threats from bacteria-induced diseases and their potential outbreak among densely populated communities, an intrinsic, low-cost biosensor device that can perform quick and precise identification of the infection type is in high demand to respond to such challenging situations and control the damage those diseases could possibly cause. This dissertation describes the development of a biosensor platform that utilizes polymer thin films, known as ionic self-assembled multilayer (ISAM) films, to be the sensitivity- enhancing medium between an LPG fiber and specific, recognition layer. With the aid of cross- linking reactions, monoclonal antibodies (IgG) or DNA probes are immobilized onto the surface of the ISAM-coated fiber, which form the core component of the biosensor. By immersing such biosensor fiber into a sample suspension, the immobilized antibody molecules will bind the specific antigen and capture the target cells or cell fragments onto the surface of the fiber sensor, resulting in increasing the average thickness of the fiber cladding and changing the refractive index of the cladding. This change occurring at the surface of the fiber results in a decrease of optical power emerging from the LPG section of the fiber. By comparing the transmitted optical power before and after applying the sample suspension, we are able to determine whether or not certain bacterial species have attached to the surface of the fiber, and as a consequence, we are able to determine whether or not the solution contains the targeted bacteria. This platform has the potential for detection of a wide range of bacteria types. In our study, we have primarily investigated the sensitivity and specificity of the biosensor to methicillin- resistant Staphlococcus aureus (MRSA). The data we obtained have shown a sensitive threshold at as low as 102 cfu/ml with pure culture samples. A typical MRSA antibody-based biosensor assay with MRSA sample at this concentration has shown optical power reduction of 21.78%. In a detailed study involving twenty-six bacterial strains possessing the PBP2a protein that enables antibiotic resistance and sixteen strains that do not, the biosensor system was able to correctly identify every sample in pure culture samples at concentration of 104 cfu/ml. Further studies have also been conducted on infected mouse tissues and clinical swab samples from human ears, noses, and skin, and in each case, the system was in full agreement with the results of standard culture tests. However, the system is not yet able to correctly distinguish MRSA and non-MRSA infections in clinical swab samples taken from infected patient wounds. It is proposed that nonspecific binding due to insufficient blocking methods is the key issue. Other bacterial strains, such as Brucella and Francisella tularensis have also been studied using a similar biosensor platform with DNA probes and antibodies, respectively, and the outcomes are also promising. The Brucella DNA biosensor is able to reflect the existence of 3 Brucella strains at 100 cfu/ml with an average of 12.2% signal reduction, while negative control samples at 106cfu/ml generate an average signal reduction of -2.1%. Similarly, the F. tularensis antibodies biosensor has shown a 25.6% signal reduction to LVS strain samples at 100 cfu/ml, while for negative control samples at the same concentration, it only produces a signal reduction of 0.05%. In general, this biosensor platform has demonstrated the potential of detecting a wide range of bacteria in a rapid and relatively inexpensive manner.
- Effect of Sialylation of Histophilus somni Lipooligosaccharide on Virulence and Resistance to Host DefensesBalyan, Rajiv (Virginia Tech, 2007-08-06)Incorporation of N-acetyl neuraminic acid (NANA), or sialic acid, onto lipooligosaccharide (LOS) enhances the virulence of several bacterial species. In the present study, we assessed the effect of sialylation of Histophilus somni LOS on complement-mediated killing, binding of complement factor H (which converts C3b to inactive C3b (iC3b) and inhibit the alternative complement pathway) to the bacteria, complement activation by the LOS, and phagocytosis and killing of the bacteria by bovine polymorphonuclear leukocytes (PMN). Killing of H. somni by alternative complement pathway was measured by incubation of sialylated or non-sialylated H. somni with antibody-free precolostral calf serum (PCS) followed by viable plate count. A complement dose-dependent response to killing of non-sialylated H. somni by PCS was observed. However, sialylated H. somni were significantly (P = 0.001) more resistant to killing at any of the concentrations of PCS used. Sialylated H. somni LOS activated (P = 0.025) and consumed (P = 0.001) less complement than non-sialylated LOS, as determined by reduction in hemolysis of opsonized sheep red blood cells or rabbit red blood cells, and by western blotting of C3 activation products. Sialylated H. somni bound more factor H than non-sialylated bacteria (determined by enzyme-linked immunosorbent assay) (P = 0.004), supporting the deficiencies observed in complement activation and consumption by sialylated LOS. Sialylation of H. somni inhibited both PMN phagocytosis of 3H-thymidine-labelled bacteria (P = 0.004) and intracellular killing of the bacteria (P = 0.0001), compared to non-sialylated bacteria. Therefore, sialylation of the LOS results in enhanced binding of complement factor H to the bacteria, resulting in diminished complement activation, resistance to complement-mediated lysis, and PMN phagocytosis and killing.
- Formation of the Francisella tularensis Biofilm is Affected by Cell Surface Glycosylation, Growth Medium, and a Glucan ExopolysaccharideChampion, Anna E.; Catanzaro, Kelly C. Freudenberger; Bandara, Aloka B.; Inzana, Thomas J. (Springer Nature, 2019-08-22)Biofilms are matrix-associated communities that enable bacteria to colonise environments unsuitable for free-living bacteria. The facultative intracellular pathogen Francisella tularensis can persist in water, amoebae, and arthropods, as well as within mammalian macrophages. F. tularensis Types A and B form poor biofilms, but F. tularensis mutants lacking lipopolysaccharide O-antigen, O-antigen capsule, and capsule-like complex formed up to 15-fold more biofilm than fully glycosylated cells. The Type B live vaccine strain was also 50% less capable of initiating surface attachment than mutants deficient in O-antigen and capsule-like complex. However, the growth medium of all strains tested also influenced the formation of biofilm, which contained a novel exopolysaccharide consisting of an amylose-like glucan. In addition, the surface polysaccharide composition of the bacterium affected the protein: DNA: polysaccharide composition of the biofilm matrix. In contrast, F. novicida attached to surfaces more efficiently and made a more robust biofilm than Type A or B strains, but loss of O-antigen or capsule-like complex did not significantly affect F. novicida biofilm formation. These results indicated that suppression of surface polysaccharides may promote biofilm formation by F. tularensis Types A and B. Whether biofilm formation enhances survival of F. tularensis in aquatic or other environmental niches has yet to be determined.
- Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane VesiclesChampion, Anna E.; Bandara, Aloka B.; Mohapatra, Nrusingh; Fulton, Kelly M.; Twine, Susan M.; Inzana, Thomas J. (Frontiers, 2018-06-15)Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylationmutant would be attenuated inmice. The CLCs isolated from LVS extracted with 0.5% phenol or 1M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/cmice, and immunization with the CLC did not protectmice against high dose respiratory challenge with type A strain SCHU S4.
- Genomic Instability and Gene Dosage Obscures Clues to Virulence Mechanisms of F. tularensis speciesModise, Thero (Virginia Tech, 2016-09-06)The pathogen Francisella tularensis subsp. tularensis has been classified as a Center for Disease Control (CDC) select agent. However, little is still known of what makes the bacteria cause dis-ease, especially the highly virulent type A1 strains. The work in this dissertation focused on type A1 strains from the Inzana laboratory, including a wildtype virulent strain TI0902, an avirulent chemical mutant strain TIGB03 with a Single Nucleotide Polymorphism in the wbtK gene, and several complemented mutants, [wbtK+]TIGB03, with dramatic differences in virulence and growth rates. One of the complemented clones (Clone12 or avp-[wbtK+]TIGB03-C12) was aviru-lent, but protected mice against challenge of a lethal dose of TI0902 and was considered as a possible vaccine strain. Whole genome sequencing was performed to identify genetic differences between the virulent, avirulent and protective strains using both Roche/454 and Illumina next-generation sequencing technologies. Additionally, RNASeq analysis was performed to identify differentially expressed genes between the different strains. This comprehensive genomic analysis revealed the critical role of transposable elements in inducing genomic instability resulting in large du-plications and deletions in the genomes of the chemical mutant and the complemented clones that in turn affect gene dosage and expression of genes known to regulate virulence. For exam-ple, whole genome sequencing of the avirulent chemical mutant TIGB03 revealed a large 75.5 kb tandem duplication flanked by transposable elements, while the genome of a virulent Clone01 (vir-[wbtK+]TIGB03-C1) lost one copy of the 75.5 kb tandem duplicated region but gained a tandem duplication of another large 80kb region that contains a virulence associated transcription factor SspA. RNAseq data showed that the dosage effect of this extra region in Clone1 suppresses expression of MglA regulated genes. Since MglA regulates genes that are known to be crucial for virulence, including the well-studied Francisella Pathogenicity Island (FPI), these results suggest that gene dosage effects arising from large duplications can trigger unknown virulence mechanisms in F. tularensis subsp. tularensis. These results are important especially when designing live vaccine strains that have repeated insertion elements in their genomes.
- Glycosylation of a Capsule-Like Complex (CLC) by Francisella novicida Is Required for Virulence and Partial Protective Immunity in MiceCatanzaro, Kelly C. Freudenberger; Champion, Anna E.; Mohapatra, Nrusingh; Cecere, Thomas E.; Inzana, Thomas J. (Frontiers, 2017-05-30)Francisella tularensis is a Gram-negative bacterium and the etiologic agent of tularemia. F. tularensis may appear encapsulated when examined by transmission electron microscopy (TEM), which is due to production of an extracellular capsule-like complex (CLC) when the bacterium is grown under specific environmental conditions. Deletion of two glycosylation genes in the live vaccine strain (LVS) results in loss of apparent CLC and attenuation of LVS in mice. In contrast, F. novicida, which is also highly virulent for mice, is reported to be non-encapsulated. However, the F. novicida genome contains a putative polysaccharide locus with homology to the CLC glycosylation locus in F. tularensis. Following daily subculture of F. novicida in Chamberlain's defined medium, an electron dense material surrounding F. novicida, similar to the F. tularensis CLC, was evident. Extraction with urea effectively removed the CLC, and compositional analysis indicated the extract contained galactose, glucose, mannose, and multiple proteins, similar to those found in the F. tularensis CLC. The same glycosylation genes deleted in LVS were targeted for deletion in F. novicida by allelic exchange using the same mutagenesis vector used for mutagenesis of LVS. In contrast, this mutation also resulted in the loss of five additional genes immediately upstream of the targeted mutation (all within the glycosylation locus), resulting in strain F. novicida Delta 1212-1218. The subcultured mutant F. novicida Delta 1212-1218 was CLC-deficient and the CLC contained significantly less carbohydrate than the subcultured parent strain. The mutant was severely attenuated in BALB/c mice inoculated intranasally, as determined by the lower number of F. novicida Delta 1212-1218 recovered in tissues compared to the parent, and by clearance of the mutant by 10-14 days post-challenge. Mice immunized intranasally with F. novicida Delta 1212-1218 were partially protected against challenge with the parent, produced significantly reduced levels of inflammatory cytokines, and their spleens contained only areas of lymphoid hyperplasia, whereas controlmice challenged with the parent exhibited hypercytokinemia and splenic necrosis. Therefore, F. novicida is capable of producing a CLC similar to that of F. tularensis, and glycosylation of the CLC contributed to F. novicida virulence and immunoprotection.
- Horizontal gene transfer in Histophilus somni and its role in the evolution of pathogenic strain 2336, as determined by comparative genomic analysesSiddaramappa, Shivakumara; Challacombe, Jean F.; Duncan, Alison J.; Gillaspy, Allison F.; Carson, Matthew; Gipson, Jenny; Orvis, Joshua; Zaitshik, Jeremy; Barnes, Gentry; Bruce, David; Chertkov, Olga; Detter, J. Chris; Han, Cliff S.; Tapia, Roxanne; Thompson, Linda S.; Dyer, David W.; Inzana, Thomas J. (2011-11-23)Background Pneumonia and myocarditis are the most commonly reported diseases due to Histophilus somni, an opportunistic pathogen of the reproductive and respiratory tracts of cattle. Thus far only a few genes involved in metabolic and virulence functions have been identified and characterized in H. somni using traditional methods. Analyses of the genome sequences of several Pasteurellaceae species have provided insights into their biology and evolution. In view of the economic and ecological importance of H. somni, the genome sequence of pneumonia strain 2336 has been determined and compared to that of commensal strain 129Pt and other members of the Pasteurellaceae. Results The chromosome of strain 2336 (2,263,857 bp) contained 1,980 protein coding genes, whereas the chromosome of strain 129Pt (2,007,700 bp) contained only 1,792 protein coding genes. Although the chromosomes of the two strains differ in size, their average GC content, gene density (total number of genes predicted on the chromosome), and percentage of sequence (number of genes) that encodes proteins were similar. The chromosomes of these strains also contained a number of discrete prophage regions and genomic islands. One of the genomic islands in strain 2336 contained genes putatively involved in copper, zinc, and tetracycline resistance. Using the genome sequence data and comparative analyses with other members of the Pasteurellaceae, several H. somni genes that may encode proteins involved in virulence (e.g., filamentous haemaggutinins, adhesins, and polysaccharide biosynthesis/modification enzymes) were identified. The two strains contained a total of 17 ORFs that encode putative glycosyltransferases and some of these ORFs had characteristic simple sequence repeats within them. Most of the genes/loci common to both the strains were located in different regions of the two chromosomes and occurred in opposite orientations, indicating genome rearrangement since their divergence from a common ancestor. Conclusions Since the genome of strain 129Pt was ~256,000 bp smaller than that of strain 2336, these genomes provide yet another paradigm for studying evolutionary gene loss and/or gain in regard to virulence repertoire and pathogenic ability. Analyses of the complete genome sequences revealed that bacteriophage- and transposon-mediated horizontal gene transfer had occurred at several loci in the chromosomes of strains 2336 and 129Pt. It appears that these mobile genetic elements have played a major role in creating genomic diversity and phenotypic variability among the two H. somni strains.
- Identification and characterization of a novel capsule-like complex surface antigen of Francisella tularensisChampion, Anna Elizabeth (Virginia Tech, 2014-12-11)Francisella tularensis is a highly virulent zoonotic pathogen that is the causative agent of tularemia in humans. Two subspecies of F. tularensis are the most virulent in humans: tularensis (type A) and holarctica (type B), with less than 10 organisms via aerosol of a type A strain having the ability to cause fatal infection. Over the last decade much research has been done on the pathogenesis of this unique intracellular bacterium and many different virulence factors have been identified. The goal of this dissertation has been to identify and characterize the capsule-like complex (CLC) surface antigen of F. tularensis, and to determine its role in virulence and immunoprotection in a mouse model. In addition, I have investigated the role of CLC in biofilm formation. The CLC appears as a negatively staining material surrounding F. tularensis cells during transmission electron microscopy (TEM). I found that the CLC in the type B live vaccine strain (LVS) could be significantly diminished by deleting two glycosyl transferase genes (LVSΔ1423-22) in the putative polysaccharide locus, FTL_1432-FTL_1421. In addition, I determined that the CLC was not a typical polysaccharide capsule, but was in fact composed of over 50 proteins and glycoproteins including known virulence determinants, such as GroEL, DnaK, and ClpB. Upon further evaluation of the CLC, I determined that it was composed of an increase in production of outer membrane vesicles and tubules (OMV/T). These OMV/T appeared to be self-aggregating into what I visualized through TEM as the CLC. LVSΔ1423-22 was attenuated in the mouse model, and BALB/c mice immunized with CLC and adjuvant were protected against challenge with LVS. In addition to virulence, the CLC appears to play a role in biofilm formation and development. F. tularensis type B strains lacking the surface antigens CLC or CLC and O-antigen, develop a 2-7-fold more robust biofilm than the parent strains. The biofilm matrix contains a glucan-like EPS, proteins, and extracellular DNA, and further characterization may lead to determining if the biofilm acts as an environmental survival mechanism for F. tularensis. In summary, the CLC appears to be a novel surface antigen composed of upregulated OMV/T that is present in type A and B F. tularensis. Deficiency in CLC contributes to increased biofilm formation that could contribute to the survival of F. tularensis in a wide range of environmental niches. Furthermore, the CLC contributes to virulence of type B strains and elicits a protective immune response to type B challenge. A CLC-deficient type A strain could be a candidate for a new live vaccine strain, and therefore further investigation of such a mutant is warranted.
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