The bovine immune response to leptospira borgpetersenii serovar hardjo

Leptospirosis, caused by the spirochaete Leptospira, is a widespread disease that affects virtually all known mammals. Pathogenic Leptospira is classified into a number of diverse species and further subdivided into over 250 serovars, based on surface antigens. As classification of Leptospira includes both antigenic and genetic systems, certain serovars, such as Hardjo, may include organisms from two different species (i.e. L. borgpetersenii serovar Hardjo subtype Hardjobovis and L. interrogans subtype Hardjoprajitno). Subtype Hardjobovis is the most common cause of bovine leptospirosis. Infection can result in mastitis, abortion and foetal death. Cattle are the maintenance host for Hardjobovis and are responsible for the spread and shed of the organism, which may result in zoonotic infections. Historically, protective immunity to Leptospira spp. was thought to be exclusively humoral, due to its serovar-specific nature and the ability to transfer passive immunity between some leptospiral animal models such as mice to guinea pigs. However, protective immunity against subtype Hardjobovis in cattle requires a cellular immune response. Vaccination of cattle with a protective subtype Hardjobovis bacterin stimulates a Th1 response characterised by IFN-γ production. Production of IFN-γ closely correlates with protective immunity in cattle. However, the antigens involved in stimulating this immune response have not been identified. The genome of L. borgpetersenii serovar Hardjo subtype Hardjobovis strain L550 has been fully sequenced. Bioinformatic analysis of the genome was used to identify 260 potential surface exposed, candidate vaccine antigens. These proteins included: predicted secreted proteins, predicted outer membrane proteins, leucine-rich repeat proteins, TPR repeat proteins, proteases, iron acquisition proteins, homologs of known bacterial virulence factors and homologs of known protective antigens in Pasteurella multocida. The list also includes 45 conserved hypothetical proteins and 103 unique hypothetical proteins. Of the 260 proteins identified, 238 proteins were successfully cloned, expressed in E. coli and purified. These proteins were then screened for their ability to stimulate a cell-mediated immune response in vaccinated cattle blood. In order to screen these proteins cattle were vaccinated with a monovalent L. borgpetersenii serovar Hardjo subtype Hardjobovis vaccine. The cattle were periodically bled and the whole blood was initially cultured with L. borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjo WCL), to determine whether the vaccine stimulated the desired immune response. Once this was established the recombinant proteins and additional potential antigens, such as whole cell sonicate fractions of Hardjo WCL were also screened for the ability to stimulate IFN-γ in vaccinated cattle blood. From this study it was found that LipL32, the major outer-membrane protein of pathogenic Leptospira spp., is involved in stimulation of IFN-γ in the blood of vaccinated cattle. Sera from the vaccinated cattle also recognised recombinant LipL32, indicating that anti-LipL32 antibodies were produced. Therefore, LipL32 can stimulate both humoral and cell-mediated immune responses in vaccinated cattle. In an attempt to identify the regions of LipL32 involved in T-cell interactions, a number of truncations were prepared and screened using the whole blood assay. Data from this study indicates that amino acids 20-106 are involved in T-cell interactions, as this peptide stimulated equal or greater levels of IFN-γ in the blood of vaccinated animals when compared to LipL32 or Hardjo WCL alone. Further characterisation of the bovine immune response to Hardjo WCL, recombinant LipL32 and the soluble master pool was also undertaken. Transcript levels of a number of Th1 and Th2 cytokine genes were measured. FACS analysis was used to determine the T-cell populations present in blood cultured with Hardjo WCL and recombinant LipL32. It has also been demonstrated that the strain of Hardjobovis used in vaccine formulation and challenge studies can affect the severity of disease. Therefore, genomic and proteomic comparative analysis of three Hardjobovis isolates was undertaken to identify any potential differences that may contribute to stimulating a cell mediated immune response. These isolates included L550, a Queensland isolate with a fully annotated and closed genome. Strain L683, an isolate from Victoria, Australia, used in the formulation of SpirovacTM and other commercial vaccines produced by Pfizer Animal Health (Australia), and strain 93U, an isolate from the United States, used in vaccine studies but was unable to protect animals from infection. Finally, a small-scale vaccine trial was performed to assess the potential for recombinant LipL32 to stimulate protective immunity in hamsters, the accepted small animal model of leptospirosis. However, due to the nature of hamster immunity towards pathogenic Leptospira spp. the rLipL32 vaccine failed to protect the hamsters from leptosp

Characterization of two novel lipopolysaccharide phosphoethanolamine transferases in Pasteurella multocida and their role in resistance to cathelicidin-2

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen Pasteurella multocida has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the P. multocida EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to P. multocida called PetG. Transcriptomic analyses indicated that petL expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo1), directly linked to lipid A in the P. multocida glycoform A LPS. In vitro assays showed that the presence of a functional petL and petK, and therefore the presence of PEtn on lipid A and Kdo1, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo1 and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera Vibrio, Bordetella, and Haemophilus. We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.Peer reviewed: YesNRC publication: Ye

The transcriptomic response ofAcinetobacter baumanniito colistin and doripenem alone and in combination in anin vitropharmacokinetics/pharmacodynamics model

L’œuvre la plus importante du burlesque mythologique en Angleterre au XVIIe siècle est de Charles Cotton et fut publiée en 1664-65 : Scarronides or Virgil Travestie. Comme son titre indique, elle avait été inspirée par Le Virgile travesty de Paul Scarron (1648-52), de son côté le travestissement français le plus important du XVIIe siècle. Mais dans cette contribution il est montré qu’on peut trouver les débuts d’une tradition anglaise du burlesque mythologique bien avant la réception de Scarron. En outre, comme expliqué en détail ici, le burlesque anglais est très différent du burlesque français de cette époque, non seulement sur le plan du style, mais aussi sur le plan de la fonction

Perturbation of the two-component signal transduction system, BprRS, results in attenuated virulence and motility defects in Burkholderia pseudomallei

Background: Burkholderia pseudomallei is the causative agent of melioidosis, a severe invasive disease of humans and animals. Initial screening of a B. pseudomallei signature-tagged mutagenesis library identified an attenuated mutant with a transposon insertion in a gene encoding the sensor component of an uncharacterised two-component signal transduction system (TCSTS), which we designated BprRS.\ud \ud Results: Single gene inactivation of either the response regulator gene (bprR) or the sensor histidine kinase gene (bprS) resulted in mutants with reduced swarming motility and reduced virulence in mice. However, a bprRS double mutant was not attenuated for virulence and displayed wild-type levels of motility. The transcriptomes of the bprS, bprR and bprRS mutants were compared with the transcriptome of the parent strain K96243. Inactivation of the entire BprRS TCSTS (bprRS double mutant) resulted in altered expression of only nine genes, including both bprR and bprS, five phage-related genes and bpss0686, encoding a putative 5, 10-methylene tetrahydromethanopterin reductase involved in one carbon metabolism. In contrast, the transcriptomes of each of the bprR and bprS single gene mutants revealed more than 70 differentially expressed genes common to both mutants, including regulatory genes and those required for flagella assembly and for the biosynthesis of the cytotoxic polyketide, malleilactone.\ud \ud Conclusions: Inactivation of the entire BprRS TCSTS did not alter virulence or motility and very few genes were differentially expressed indicating that the definitive BprRS regulon is relatively small. However, loss of a single component, either the sensor histidine kinase BprS or its cognate response regulator BprR, resulted in significant transcriptomic and phenotypic differences from the wild-type strain. We hypothesize that the dramatically altered phenotypes of these single mutants are the result of cross-regulation with one or more other TCSTSs and concomitant dysregulation of other key regulatory genes

Novel cassette assay to quantify the outer membrane permeability of five β-lactams simultaneously in carbapenem-resistant klebsiella pneumoniae and enterobacter cloacae

Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae. Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Both clinical isolates harbored a blaKPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately (“discrete assay”) and simultaneously (“cassette assay”) by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae. Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics