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1.  Induction of the Yersinia pestis PhoP-PhoQ Regulatory System in the Flea and Its Role in Producing a Transmissible Infection 
Journal of Bacteriology  2013;195(9):1920-1930.
Transmission of Yersinia pestis is greatly enhanced after it forms a bacterial biofilm in the foregut of the flea vector that interferes with normal blood feeding. Here we report that the ability to produce a normal foregut-blocking infection depends on induction of the Y. pestis PhoP-PhoQ two-component regulatory system in the flea. Y. pestis phoP-negative mutants achieved normal infection rates and bacterial loads in the flea midgut but produced a less cohesive biofilm both in vitro and in the flea and had a greatly reduced ability to localize to and block the flea foregut. Thus, not only is the PhoP-PhoQ system induced in the flea gut environment, but also this induction is required to produce a normal transmissible infection. The altered biofilm phenotype in the flea was not due to lack of PhoPQ-dependent or PmrAB-dependent addition of aminoarabinose to the Y. pestis lipid A, because an aminoarabinose-deficient mutant that is highly sensitive to cationic antimicrobial peptides had a normal phenotype in the flea digestive tract. In addition to enhancing transmissibility, induction of the PhoP-PhoQ system in the arthropod vector prior to transmission may preadapt Y. pestis to resist the initial encounter with the mammalian innate immune response.
PMCID: PMC3624595  PMID: 23435973
2.  In Vivo Manipulation of γ9+ T Cells in the Common Marmoset (Callithrix Jacchus) with Phosphoantigen and Effect on the Progression of Respiratory Melioidosis 
PLoS ONE  2013;8(9):e74789.
Burkholderia pseudomallei is a dangerous human pathogen. Phosphoantigens specifically the target primate specific γ9+δ2+ T cells subset and some have been developed as potential immunotherapeutics. Previously, we demonstrated that, when stimulated with the phosphoantigen CHDMAPP, γ9+δ2+ T cells aid in the killing of intracellular B. pseudomallei bacteria. Moreover, we found that common marmoset (Callithrix Jacchus) γ9+ T cells increase in frequency and respond to the phosphoantigen CHDMAPP and/or B. pseudomallei, in combination with IL-2, in a similar manner to human γ9+δ2+ T cells. Here we evaluate the efficacy of the phosphoantigen CHDMAPP, in combination with IL-2, as a therapy against B. pseudomallei infection, in vivo. We found that the previous studies predicted the in vivo responsiveness of γ9+ T cells to the CHDMAPP+IL-2 treatment and significant expansion of the numbers of peripheral and splenic γ9+ T cells were observed. This effect was similar to those reported in other primate species treated with phosphoantigen. Furthermore, splenocytes were retrieved 7 days post onset of treatment, restimulated with CHDMAPP or heat-killed B. pseudomallei and the cultured γ9+ T cells demonstrated no reduction in IFN-γ response when CHDMAPP+IL-2 animals were compared to IL-2 only treated animals. Using an established model of B. pseudomallei infection in the marmoset, we assessed the potential for using phosphoantigen as a novel immunotherapy. The CHDMAPP treatment regime had no effect on the progression of respiratory melioidosis and this was despite the presence of elevated numbers of γ9+ T cells in the spleen, liver and lung and an increased proportion of IFN-γ+ cells in response to infection. We therefore report that the common marmoset has proven a good model for studying the effect in vivo of γ9+ T cell stimulation; however, γ9+ T cells have little or no effect on the progression of lethal, respiratory B. pseudomallei infection.
PMCID: PMC3786980  PMID: 24098670
3.  Structure and function of cytidine monophosphate kinase from Yersinia pseudotuberculosis, essential for virulence but not for survival 
Open Biology  2012;2(12):120142.
The need for new antibiotics has become pressing in light of the emergence of antibiotic-resistant strains of human pathogens. Yersinia pestis, the causative agent of plague, is a public health threat and also an agent of concern in biodefence. It is a recently emerged clonal derivative of the enteric pathogen Yersinia pseudotuberculosis. Previously, we developed a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. One such target was cytidine monophosphate (CMP) kinase, which is an essential gene in some organisms. Previously, we had thought CMP kinase was essential for Y. pseudotuberculosis, but by modification of the mutagenesis approach, we report here the production and characterization of a Δcmk mutant. The isogenic mutant had a growth defect relative to the parental strain, and was highly attenuated in mice. We have also elucidated the structure of the CMP kinase to 2.32 Å, and identified three key residues in the active site that are essential for activity of the enzyme. These findings will have implications for the development of novel CMP kinase inhibitors for therapeutic use.
PMCID: PMC3603445  PMID: 23271832
cytidine monophosphate kinase; crystal structure; virulence; Yersinia
4.  O-Linked Glycosylation of the PilA Pilin Protein of Francisella tularensis: Identification of the Endogenous Protein-Targeting Oligosaccharyltransferase and Characterization of the Native Oligosaccharide▿† 
Journal of Bacteriology  2011;193(19):5487-5497.
Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.
PMCID: PMC3187425  PMID: 21804002
5.  Glycosylation of DsbA in Francisella tularensis subsp. tularensis▿† 
Journal of Bacteriology  2011;193(19):5498-5509.
In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis.
PMCID: PMC3187430  PMID: 21803994
6.  A Yersinia pestis guaBA mutant is attenuated in virulence and provides protection against plague in a mouse model of infection 
Microbial pathogenesis  2010;48(5):191-195.
There is a need to develop effective countermeasures for Yersinia pestis, the etiologic agent of plague and a potential bioterrorism agent. Salmonella and Shigella spp. deleted in the guaBA genes involved in guanine biosynthesis have been shown to be attenuated in vivo. In this study, we sought to determine whether deletion of the guaBA operon would render Y. pestis auxotrophic for guanine and avirulent; such a strain could serve as a live attenuated plague vaccine candidate. A Y. pestis guaBA mutant was generated by specific deletion of a segment of the guaBA operon, producing a guanine auxotroph that was highly attenuated in a mouse model of Y. pestis infection. Furthermore, mice vaccinated with a single dose of 7×104 CFU via the intravenous route were fully protected against subsequent lethal challenge with the Y. pestis parental strain. These findings identify guaBA as a target for deletion to generate a live attenuated plague vaccine.
PMCID: PMC2857377  PMID: 20096773
Yersinia pestis; plague; guanine; mutant; live attenuated vaccines
7.  Molecular Recognition of Chymotrypsin by the Serine Protease Inhibitor Ecotin from Yersinia pestis* 
The Journal of Biological Chemistry  2011;286(27):24015-24022.
Resistance to antibiotics is a problem not only in terms of healthcare but also biodefense. Engineering of resistance into a human pathogen could create an untreatable biothreat pathogen. One such pathogen is Yersinia pestis, the causative agent of plague. Previously, we have used a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. The serine protease inhibitor ecotin was identified as one such target. We have carried out mutational analyses in the closely related Yersinia pseudotuberculosis, validating that the ecotin gene is a virulence-associated gene in this bacterium. Y. pestis ecotin inhibits chymotrypsin. Here, we present the structure of ecotin in complex with chymotrypsin to 2.74 Å resolution. The structure features a biologically relevant tetramer whereby an ecotin dimer binds to two chymotrypsin molecules, similar to what was observed in related serine protease inhibitor structures. However, the vast majority of the interactions in the present structure are distinctive, indicating that the broad specificity of the inhibitor for these proteases is based largely on its capacity to recognize features unique to each of them. These findings will have implications for the development of small ecotin inhibitors for therapeutic use.
PMCID: PMC3129183  PMID: 21531711
Crystallography; Enzyme Structure; Protease; Protease Inhibitor; Protein-Protein Interactions; Molecular Recognition
8.  Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization 
Vaccine  2009;28(7):1824.
Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and Intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4ΔclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4ΔclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Δ0918ΔcapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4ΔclpB, or SCHU S4Δ0918ΔcapB provided no obvious correlate to their relative efficacies.
PMCID: PMC2822029  PMID: 20018266
9.  Kinetic Analysis of Yersinia pestis DNA Adenine Methyltransferase Activity Using a Hemimethylated Molecular Break Light Oligonucleotide 
PLoS ONE  2007;2(8):e801.
DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam) has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics.
Methodology/Principal Findings
Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein) and quencher (dabcyl) and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71±0.07 indicating that it is a sensitive assay for the identification of inhibitors.
The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.
PMCID: PMC1949145  PMID: 17726531
10.  The Immunologically Distinct O Antigens from Francisella tularensis Subspecies tularensis and Francisella novicida Are both Virulence Determinants and Protective Antigens▿  
Infection and Immunity  2006;75(1):371-378.
We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.
PMCID: PMC1828428  PMID: 17074846
11.  Pathogenesis of Yersinia pestis Infection in BALB/c Mice: Effects on Host Macrophages and Neutrophils  
Infection and Immunity  2005;73(11):7142-7150.
The pathogenesis of infection with Yersinia pestis, the causative agent of plague, was examined following subcutaneous infection of BALB/c mice with a fully virulent strain expressing green fluorescent protein. Plate culturing, flow cytometry, and laser confocal microscopy of spleen homogenates throughout infection revealed three discernible stages of infection. The early phase was characterized by the presence of a small number of intracellular bacteria mostly within CD11b+ macrophages and Ly-6G+ neutrophils. These bacteria were not viable, as determined by plate culturing of spleen homogenates, until day 2 postinfection. Between days 2 and 4 postinfection, a plateau phase was observed, with bacterial burdens of 103 to 104 CFU per spleen. Flow cytometric analysis revealed that there was even distribution of Y. pestis within both CD11b+ macrophage and Ly-6G+ neutrophil populations on day 2 postinfection. However, from day 3 postinfection onward, intracellular bacteria were observed exclusively within splenic CD11b+ macrophages. The late phase of infection, between days 4 and 5 postinfection, was characterized by a rapid increase in bacterial numbers, as well as escape of bacteria into the extracellular compartment. Annexin V staining of spleens indicated that a large proportion of splenic neutrophils underwent rapid apoptosis on days 1 and 2 postinfection. Fewer macrophages underwent apoptosis during the same period. Our data suggest that during the early stages of Y. pestis infection, splenic neutrophils are responsible for limiting the growth of Y. pestis and that splenic macrophages provide safe intracellular shelters within which Y. pestis is able to grow and escape during the later stages of infection. This macrophage compliance can be overcome in vitro by stimulation with a combination of gamma interferon and tumor necrosis factor alpha.
PMCID: PMC1273833  PMID: 16239508
12.  Tularemia 
Clinical Microbiology Reviews  2002;15(4):631-646.
Francisella tularensis is the etiological agent of tularemia, a serious and occasionally fatal disease of humans and animals. In humans, ulceroglandular tularemia is the most common form of the disease and is usually a consequence of a bite from an arthropod vector which has previously fed on an infected animal. The pneumonic form of the disease occurs rarely but is the likely form of the disease should this bacterium be used as a bioterrorism agent. The diagnosis of disease is not straightforward. F. tularensis is difficult to culture, and the handling of this bacterium poses a significant risk of infection to laboratory personnel. Enzyme-linked immunosorbent assay- and PCR-based methods have been used to detect bacteria in clinical samples, but these methods have not been adequately evaluated for the diagnosis of pneumonic tularemia. Little is known about the virulence mechanisms of F. tularensis, though there is a large body of evidence indicating that it is an intracellular pathogen, surviving mainly in macrophages. An unlicensed live attenuated vaccine is available, which does appear to offer protection against ulceroglandular and pneumonic tularemia. Although an improved vaccine against tularemia is highly desirable, attempts to devise such a vaccine have been limited by the inability to construct defined allelic replacement mutants and by the lack of information on the mechanisms of virulence of F. tularensis. In the absence of a licensed vaccine, aminoglycoside antibiotics play a key role in the prevention and treatment of tularemia.
PMCID: PMC126859  PMID: 12364373
13.  Yersinia pestis pFra Shows Biovar-Specific Differences and Recent Common Ancestry with a Salmonella enterica Serovar Typhi Plasmid 
Journal of Bacteriology  2001;183(8):2586-2594.
Population genetic studies suggest that Yersinia pestis, the cause of plague, is a clonal pathogen that has recently emerged from Yersinia pseudotuberculosis. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of Y. pestis-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different Y. pestis strains which distinguish Y. pestis bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between Y. pestis and the exclusively human pathogen Salmonella enterica serovar Typhi.
PMCID: PMC95176  PMID: 11274119
14.  The Response Regulator PhoP Is Important for Survival under Conditions of Macrophage-Induced Stress and Virulence in Yersinia pestis 
Infection and Immunity  2000;68(6):3419-3425.
The two-component regulatory system PhoPQ has been identified in many bacterial species. However, the role of PhoPQ in regulating virulence gene expression in pathogenic bacteria has been characterized only in Salmonella species. We have identified, cloned, and sequenced PhoP orthologues from Yersinia pestis, Yersinia pseudotuberculosis, and Yersinia enterocolitica. To investigate the role of PhoP in the pathogenicity of Y. pestis, an isogenic phoP mutant was constructed by using a reverse-genetics PCR-based strategy. The protein profiles of the wild-type and phoP mutant strains, grown at either 28 or 37°C, revealed more than 20 differences, indicating that PhoP has pleiotrophic effects on gene expression in Y. pestis. The mutant showed a reduced ability to survive in J774 macrophage cell cultures and under conditions of low pH and oxidative stress in vitro. The mean lethal dose of the phoP mutant in mice was increased 75-fold in comparison with that of the wild-type strain, indicating that the PhoPQ system plays a key role in regulating the virulence of Y. pestis.
PMCID: PMC97616  PMID: 10816493

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