Aloka B. Bandara

Functional Analysis of a Predicted Flavonol Synthase Gene Family in Arabidopsis

The genome of Arabidopsis (Arabidopsis thaliana) contains five sequences with high similarity to FLAVONOL SYNTHASE1 (AtFLS1), a previously characterized flavonol synthase gene that plays a central role in flavonoid metabolism. This apparent redundancy suggests the possibility that Arabidopsis uses multiple isoforms of FLS with different substrate specificities to mediate the production of the flavonols, quercetin and kaempferol, in a tissue-specific and inducible manner. However, biochemical and genetic analysis of the six AtFLS sequences indicates that, although several of the members are expressed, only AtFLS1 encodes a catalytically competent protein. AtFLS1 also appears to be the only member of this group that influences flavonoid levels and the root gravitropic response in seedlings under nonstressed conditions. This study showed that the other expressed AtFLS sequences have tissue- and cell type-specific promoter activities that overlap with those of AtFLS1 and encode proteins that interact with other flavonoid enzymes in yeast two-hybrid assays. Thus, it is possible that these “pseudogenes” have alternative, noncatalytic functions that have not yet been uncovered.

Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice

BackgroundIn prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure 1 and ure 2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing.ResultsThe deduced amino acid sequence of urease-α subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Δure 1K (generated by deleting ureD and ureA in ure 1 operon), strain 1330Δure 2K (ureB and ureC in ure 2 operon), strain 1330Δure 2C (ureA, ureB, and ureC in ure 2 operon), and strain 1330Δure 1KΔure 2C (ureD and ureA in ure 1 operon and ureA, ureB, and ureC in ure 2 operon). When grown in urease test broth, strains 1330, 1330Δure 2K and 1330Δure 2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Δure 1K and 1330Δure 1KΔure 2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Δure 1K and 1330Δure 1KΔure 2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Δure 1KΔure 2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Δure 1K was completely killed, strain 1330Δure 2C was partially killed, but strains 1330 and 1330Δure 2K were not killed.ConclusionThese findings suggest that the ure 1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure 2 operon apparently enhances the resistance to low-pH killing in-vitro.

Carboxyl-Terminal Protease Regulates Brucella suis Morphology in Culture and Persistence in Macrophages and Mice

The putative carboxyl-terminal processing protease (CtpA) of Brucella suis 1330 is a member of a novel family of endoproteases involved in the maturation of proteins destined for the cell envelope. The B. suis CtpA protein shared up to 77% homology with CtpA proteins of other bacteria. A CtpA-deficient Brucella strain (1330DeltactpA), generated by allelic exchange, produced smaller colonies on enriched agar plates and exhibited a 50% decrease in growth rate in enriched liquid medium and no growth in salt-free enriched medium compared to the wild-type strain 1330 or the ctpA-complemented strain 1330DeltactpA[pBBctpA]. Electron microscopy revealed that in contrast to the native coccobacillus shape of wild-type strain 1330, strain 1330DeltactpA possessed a spherical shape, an increased cell diameter, and cell membranes partially dissociated from the cell envelope. In the J774 mouse macrophage cell line, 24 h after infection, the CFU of the strain 1330DeltactpA declined by approximately 3 log(10) CFU relative to wild-type strain 1330. Nine weeks after intraperitoneal inoculation of BALB/c mice, strain 1330DeltactpA had cleared from spleens but strain 1330 was still present. These observations suggest that the CtpA activity is necessary for the intracellular survival of B. suis. Relative to the saline-injected mice, strain 1330DeltactpA-vaccinated mice exhibited 4 to 5 log(10) CFU of protection against challenge with virulent B. abortus strain 2308 or B. suis strain 1330 but no protection against B. melitensis strain 16 M. This is the first report correlating a CtpA deficiency with cell morphology and attenuation of B. suis.

Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

ABSTRACT The capsular polysaccharide (CP) of Actinobacillus pleuropneumoniae is required for virulence of the bacteria in swine. However, a molecular investigation of whether the type or quantity of CP affects A. pleuropneumoniae virulence has not been reported. To initiate this investigation, a DNA region downstream of conserved genes required for CP export in A. pleuropneumoniae serotype 1 was cloned and sequenced. Three open reading frames, designated cps1A, cps1B, and cps1C, were identified that had amino acid homology to bacterial carbohydrate biosynthesis genes. A kanamycin resistance cassette (Kanr) was inserted into a 750-bp deletion spanning cps1AB or into a 512-bp deletion in cps1B only, and the constructs were cloned in a suicide vector. The Kanr gene was then transferred into the chromosome of strain 4074 by homologous recombination to produce strain 4074Δcps1N and strain 4074Δcps1B, respectively. Strain 4074Δcps1N produced no detectable CP, but strain 4074Δcps1B made 15% of the serotype 1 CP made by the parent strain, 4074, as determined by enzyme-linked immunosorbent assay and precipitation of free CP. The cps1ABC genes of strain 4074 and the cps5ABC and cps5ABCDE genes of serotype 5a strain J45 were cloned into the shuttle vector pLS88 and electroporated into 4074Δcps1N to produce 4074Δcps1N(pABcps101), 4074Δcps1N(pJMLcps53), and 4074Δcps1N(pABcps55), respectively. Strain 4074Δcps1N(pABcps101) produced about 33% of the serotype 1 CP produced by strain 4074. Strains 4074Δcps1N(pJMLcps53) and 4074Δcps1N(pABcps55) produced serotype 5a CP in similar quantity or in fourfold excess, respectively, to that produced by strain 4074. With intratracheal challenge in pigs at similar dosages, the order of virulence of strains producing serotype 1 CP (assessed by mortality, lung consolidation, hemorrhage, and fibrinous pleuritis) was the following: strain 4074 > strain 4074Δcps1N(pABcps101) ≥ strain 4074Δcps1N > strain 4074Δcps1B. Strain 4074Δcps1N(pJMLcps53) was less virulent than strain 4074Δcps1N(pABcps55). However, both strains produced serotype 5a CP in similar or greater quantities than was observed for production of serotype 1 CP by the parent strain, 4074, but were less virulent than the parent strain. Therefore, the amount of serotype 1 or 5a CP produced by isogenic strains of A. pleuropneumoniae correlated with the virulence of the bacteria in pigs. However, virulence was also influenced by the type of CP produced or by its mechanism of expression.

Photonic Biosensor Assays to Detect and Distinguish Subspecies of Francisella tularensis

The application of photonic biosensor assays to diagnose the category-A select agent Francisella tularensis was investigated. Both interferometric and long period fiber grating sensing structures were successfully demonstrated; both these sensors are capable of detecting the optical changes induced by either immunological binding or DNA hybridization. Detection was made possible by the attachment of DNA probes or immunoglobulins (IgG) directly to the fiber surface via layer-by-layer electrostatic self-assembly. An optical fiber biosensor was tested using a standard transmission mode long period fiber grating of length 15 mm and period 260 μm, and coated with the IgG fraction of antiserum to F. tularensis. The IgG was deposited onto the optical fiber surface in a nanostructured film, and the resulting refractive index change was measured using spectroscopic ellipsometry. The presence of F. tularensis was detected from the decrease of peak wavelength caused by binding of specific antigen. Detection and differentiation of F. tularensis subspecies tularensis (type A strain TI0902) and subspecies holarctica (type B strain LVS) was further accomplished using a single-mode multi-cavity fiber Fabry-Perot interferometric sensor. These sensors were prepared by depositing seven polymer bilayers onto the fiber tip followed by attaching one of two DNA probes: (a) a 101-bp probe from the yhhW gene unique to type-A strains, or (b) a 117-bp probe of the lpnA gene, common to both type-A and type-B strains. The yhhW probe was reactive with the type-A, but not the type-B strain. Probe lpnA was reactive with both type-A and type-B strains. Nanogram quantities of the target DNA could be detected, highlighting the sensitivity of this method for DNA detection without the use of PCR. The DNA probe reacted with 100% homologous target DNA, but did not react with sequences containing 2-bp mismatches, indicating the high specificity of the assay. These assays will fill an important void that exists for rapid, culture-free, and field-compatible diagnosis of F. tularensis.

Incidence of brucellosis in Sri Lanka: an overview

Infection by Brucella abortus seems to be a major cause of abortions among cattle and buffaloes in Sri Lanka. The incidence of this disease is more prominent among the animals in the Dry zone of the country raised under extensive management systems. The present low incidence of this disease and the small size of the country may facilitate launching of an effective disease control scheme. The milk ring test (MRT) has proven to be usable in testing milk for the infection at farm level. An ELISA technique could be employed to test the seroprevalence of infection among MRT-positive animals. A program to purchase the diseased animals by the state for slaughter, and a countrywide vaccination program with B. abortus strain RB51 would enable the countrys livestock industry to eventually eradicate this disease.

The putative penicillin-binding proteins 1 and 2 are important for viability, growth and cell morphology of Brucella melitensis.

The penicillin-binding proteins (PBPs) are enzymes that regulate the assembly of the peptidoglycan layer of the bacterial cell wall. The genome of Brucella melitensis strain 16M possesses seven pbp genes: three in pbp-1 family (designated as 1A, 1B, and 1C); one in pbp-2 family; and three in pbp-6 family (designated as 6A, 6B, and 6C). We investigated the importance of pbp-1 and pbp-2 genes to viability, cell morphology and infectivity of B. melitensis. A recombinant B. melitensis strain (designated 16MDeltapbp1C) was generated by disrupting the pbp-1C of strain 16M by allelic exchange. This strain produced nearly 20% smaller colonies on trypticase soy agar plates, and grew slower in trypticase soy broth compared to the strain 16M. Electron microscopy revealed that strain 16M exhibited native cocco-bacillus morphology, while 16MDeltapbp1C possessed a spherical morphology. Strain 16MDeltapbp1C did not differ from strain 16M in terms of recovery from infected mouse macrophage cell line J774.1, or recovery from spleens of infected BALB/c mice, suggesting that pbp-1C is dispensable for intracellular persistence of B. melitensis. Expression of mRNA of fixR, the gene downstream of pbp-1C was similar between the strains 16M and 16MDeltapbp1C suggesting that disruption of pbp-1C did not induce any polar effects. Multiple attempts to mutate pbp-1A, pbp-1B, or pbp-2 genes failed, most probably because these genes are indispensable for viability of B. melitensis. Our findings suggest that pbp-1C regulates in vitro growth and cell morphology, whereas pbp-1A, pbp-1B, and pbp-2 are essential for viability of B. melitensis.

Identification of Histophilus somni by a nanomaterial optical fiber biosensor assay

Histophilus somni is an opportunistic pathogen responsible for respiratory and systemic diseases of cattle and sheep. Rapid and accurate detection of H. somni is essential to distinguish H. somni from other potential pathogens for proper control and treatment of infections. Nanomaterial optical fiber biosensors (NOFS) recognize analyte interactions, such as DNA hybridization, with high specificity and sensitivity, and were applied to detect H. somni DNA in culture and clinical samples. An ionic self-assembled multilayer (ISAM) film was fabricated on a long-period grating optical fiber, and a biotinylated, nucleotide probe complementary to the H. somni 16S rDNA gene was coupled to the ISAM film. Exposure of the ISAM::probe to ⩾100 killed cells of H. somni strain 2336 without DNA amplification resulted in attenuation of light transmission of ⩾9.4%. Exposure of the complexed fiber to Escherichia coli or non–H. somni species of Pasteurellaceae reduced light transmission by ⩽3.4%. Exposure of the ISAM::probe to blood, bronchoalveolar fluid, or spleen from mice or calves infected with H. somni resulted in ⩾24.3% transmission attenuation. The assay correctly detected all 6 strains of H. somni tested from culture, or tissues from 3 separate mice and calves tested in duplicate. Six heterologous strains (representing 6 genera) reacted at below the cutoff value of 4.87% attenuation of light transmission. NOFS detected at least 100 H. somni cells without DNA amplification within 45 min with high specificity. Although different fibers could vary in signal sensitivity, this did not affect the sensitivity or specificity of the assay.

Further characterization of the capsule-like complex (CLC) produced by Francisella tularensis subspecies tularensis: protective efficacy and similarity to outer membrane vesicles

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 glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M 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 1 M 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/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.