bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence Analysis of the
mip
Gene of the Soilborne Pathogen
Legionella longbeachae
Authors: Doyle Robyn M. , Steele Trevor W. , McLennan Alan M. , Parkinson Ian H. , Manning Paul A. , Heuzenroeder Michael W.
Published: December 2019

To understand the basis of pathogenesis by
Legionella longbeachae
serogroup 1, the importance of the Mip protein in this species was examined. Amino-terminal analysis of the purified, cloned
L. longbeachae
serogroup 1 ATCC 33462 Mip protein confirmed that the cloned gene protein was expressed and processed in an
Escherichia coli
background. DNA sequence analysis of plasmid pIMVS27, containing the entire
L. longbeachae
serogroup 1
mip
gene, revealed a high degree of homology to the
mip
gene of
Legionella pneumophila
serogroup 1, 76% homology at the DNA level and 87% identity at the amino acid level. Primer extension analysis determined that the start site of transcription was the same for both species, with some differences observed for the −10 and −35 promoter regions. Primers designed from the
mip
gene sequence obtained for
L. longbeachae
serogroup 1 ATCC 33462 were used to amplify the
mip
genes from
L. longbeachae
serogroup 2 ATCC 33484 and an Australian clinical isolate of
L. longbeachae
serogroup 1 A5H5. The
mip
gene from A5H5 was 100% identical to the type strain sequence. The serogroup 2 strain of
L. longbeachae
differed by 2 base pairs in third-codon positions. Allelic exchange mutagenesis was used to generate an isogenic
mip
mutant in ATCC 33462 and strain A5H5. The ATCC
mip
mutant was unable to infect a strain of
Acanthamoebae
sp. both in liquid and in a potting mix coculture system, while the A5H5
mip
mutant behaved in a manner siilar to that of
L. pneumophila
serogroup 1, i.e., it displayed a reduced capacity to infect and multiply within
Acanthamoebae
. To determine if this mutation resulted in reduced virulence in the guinea pig animal model, the A5H5
mip
mutant and its parent strain were assessed for their abilities to establish an infection after aerosol exposure. Unlike the virulent parent strain, the mutant strain did not kill any animals under two different dose regimes. The data indicate that the Mip protein plays an important role in the intracellular life cycle of
L. longbeachae
serogroup 1 species and is required for full virulence.
Phlebotomus papatasiSaliva Inhibits Protein Phosphatase Activity and Nitric Oxide Production by Murine Macrophages
Authors: Waitumbi John , Warburg Alon
Published: December 2019

Leishmaniaparasites, transmitted by phlebotomine sand flies, are obligate intracellular parasites of macrophages. The sand flyPhlebotomus papatasiis the vector ofLeishmania major, a causative agent of cutaneous leishmaniasis in the Old World, and its saliva exacerbates parasite proliferation and lesion growth in experimental cutaneous leishmaniasis. Here we show thatP. papatasisaliva contains a potent inhibitor of protein phosphatase 1 and protein phosphatase 2A of murine macrophages. We further demonstrate thatP. papatasisaliva down regulates expression of the inducible nitric oxide synthase gene and reduces nitric oxide production in murine macrophages. Partial biochemical characterization of the protein phosphatase and nitric oxide inhibitor indicated that it is a small, ethanol-soluble molecule resistant to boiling, proteolysis, and DNase and RNase treatments. We suggest that theP. papatasisalivary protein phosphatase inhibitor interferes with the ability of activated macrophages to transmit signals to the nucleus, thereby preventing up regulation of the induced nitric oxide synthase gene and inhibiting the production of nitric oxide. Since nitric oxide is toxic to intracellular parasites, the salivary protein phosphatase inhibitor may be the mechanism by whichP. papatasisaliva exacerbates cutaneous leishmaniasis.
Antigen-Specific CD8+T Cells Protect against Lethal Toxoplasmosis in Mice Infected withNeospora caninum
Authors: Kasper Lloyd H. , Khan Imtiaz A.
Published: December 2019

Neospora caninumis a coccidial protozoan parasite that appears morphologically indistinguishable fromToxoplasma gondiiand that infects a large range of mammals. Both inbred and outbred strains of mice exhibit a high degree of resistance to infection withN. caninum. Three inbred strains of mice (A/J, BALB/c, and C57BL/6) that were infected intraperitoneally withN. caninumwere protected against a lethal challenge fromT. gondii. Vaccine-induced protection wasNeosporadose dependent. A rise in the CD8+T-cell population in mice that had been vaccinated withN. caninumand challenged withT. gondiiwas observed. Adoptive transfer of CD8+T-cell splenocytes fromN. caninum-infected mice was protective against challenge withToxoplasma. The CD8+T cells fromNeospora-infected mice proliferate to bothNeosporaandToxoplasmaantigens in vitro and secrete substantial quantities of gamma interferon when pulsed with the parasite antigen. These observations demonstrate thatN. caninumprotects against lethalT. gondiiinfection by the induction of CD8+T cells that are immunoreactive to both parasites.
Characterization of Leptospiral Outer Membrane Lipoprotein LipL36: Downregulation Associated with Late-Log-Phase Growth and Mammalian Infection
Authors: Haake David A. , Martinich Carleen , Summers Theresa A. , Shang Ellen S. , Pruetz Jay D. , McCoy Adam M. , Mazel Mary K. , Bolin Carole A.
Published: December 2019

We report the cloning of the gene encoding a 36-kDa leptospiral outer membrane lipoprotein, designated LipL36. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment in order to design an oligonucleotide probe. A Lambda-Zap II library containingEcoRI fragments ofLeptospira kirschneriDNA was screened, and a 2.3-kb DNA fragment which contained the entire structurallipL36gene was identified. Several lines of evidence indicate that LipL36 is lipid modified in a manner similar to that of LipL41, a leptospiral outer membrane lipoprotein we described in a previous study (E. S. Shang, T. A. Summers, and D. A. Haake, Infect. Immun. 64:2322–2330, 1996). The deduced amino acid sequence of LipL36 would constitute a 364-amino-acid polypeptide with a 20-amino-acid signal peptide, followed by an L-X-Y-C lipoprotein signal peptidase cleavage site. LipL36 is solubilized by Triton X-114 extraction ofL. kirschneri; phase separation results in partitioning of LipL36 exclusively into the hydrophobic, detergent phase. LipL36 is intrinsically labeled during incubation ofL. kirschneriin media containing [3H]palmitate. Processing of LipL36 is inhibited by globomycin, a selective inhibitor of lipoprotein signal peptidase. After processing, LipL36 is exported to the outer membrane along with LipL41 and lipopolysaccharide. Unlike LipL41, there appears to be differential expression of LipL36. In early-log-phase cultures, LipL36 is one of the most abundantL. kirschneriproteins. However, LipL36 levels drop considerably beginning in mid-log phase. LipL36 expression in vivo was evaluated by examining the humoral immune response to leptospiral antigens in the hamster model of leptospirosis. Hamsters surviving challenge with culture-adapted virulentL. kirschnerigenerate a strong antibody response to LipL36. In contrast, sera from hamsters surviving challenge with host-adaptedL. kirschnerido not recognize LipL36. These findings suggest that LipL36 expression is downregulated during mammalian infection, providing a marker for studying the mechanisms by which pathogenicLeptospiraspecies adapt to the host environment.
Purification and Characterization of a Cytotoxic Exolipid of
Burkholderia pseudomallei
Authors: Häußler S. , Nimtz M. , Domke T. , Wray V. , Steinmetz I.
Published: December 2019

Burkholderia pseudomallei
is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of
B. pseudomallei
. After cultivation of
B. pseudomallei
in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-
O
-α-
l
-rhamnopyranosyl-α-
l
-rhamnopyranosyl-β-hydroxytetradecanoyl-β-hydroxytetradecanoate (Rha-Rha-C
14
-C
14
), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.
Expressed Sequence Tag Analysis of the Bradyzoite Stage of
Toxoplasma gondii
: Identification of Developmentally Regulated Genes
Authors: Manger Ian D. , Hehl Adrian , Parmley Steve , Sibley L. David , Marra Marco , Hillier Ladeana , Waterston Robert , Boothroyd John C.
Published: December 2019

Toxoplasma gondii
is a protozoan parasite responsible for widespread infections in humans and animals. Two major asexual forms are produced during the life cycle of this parasite: the rapidly dividing tachyzoite and the more slowly dividing, encysted bradyzoite. To further study the differentiation between these two forms, we have generated a large number of expressed sequence tags (ESTs) from both asexual stages. Previously, we obtained data on ∼7,400 ESTs from tachyzoites (J. Ajioka et al., Genome Res. 8:18–28, 1998). Here, we report the results from analysis of ∼2,500 ESTs from bradyzoites purified from the cysts of infected mice. We also report the results from analysis of 760 ESTs from parasites induced to differentiate from tachyzoites to bradyzoites in vitro. Comparison of the data sets from bradyzoites and tachyzoites reveals many previously uncharacterized sequence clusters which are largely or completely specific to one or other developmental stage. This class includes a bradyzoite-specific form of enolase. Combined with the previously identified bradyzoite-specific form of lactate dehydrogenase, this finding suggests significant differences in flux through the lower end of the glycolytic pathway in this stage. Thus, the generation of this data set provides valuable insights into the metabolism and growth of the parasite in the encysted form and represents a substantial body of information for further study of development in
Toxoplasma.
Local Chemokine Paralysis, a Novel Pathogenic Mechanism forPorphyromonas gingivalis
Authors: Darveau Richard P. , Belton Carol M. , Reife Robert A. , Lamont Richard J.
Published: December 2019

Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated withPorphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by bothP. gingivalisand commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast,P. gingivaliswas found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion byP. gingivalis. Furthermore, a noninvasive mutant ofP. gingivaliswas unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites ofP. gingivaliscolonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.
Adequate Expression of Protective Immunity in the Absence of Granuloma Formation inMycobacterium tuberculosis-Infected Mice with a Disruption in the Intracellular Adhesion Molecule 1 Gene
Authors: Johnson Christine M. , Cooper Andrea M. , Frank Anthony A. , Orme Ian M.
Published: December 2019

It remains unknown whether the expression of cell-mediated protective immunity and the capacity to mount a delayed-type hypersensitivity (DTH) reaction in tuberculosis infection represent two manifestations of a basic response or are dissociable events. In this study, we present data in favor of the latter hypothesis, by showing that tuberculosis infection in the lungs of mice possessing only a truncated form of intracellular adhesion molecule 1 due to gene disruption was still adequately controlled by the expression of protective immunity in the absence of any sustained influx of macrophages and the lack of formation of appreciable granulomas. These animals also had no detectable DTH response to mycobacterial proteins in the footpad assay, indicating that the accumulation of blood-borne macrophages at sites of mycobacterial infection or antigen deposition is not essential to control of the infection. These data support the hypothesis that the DTH component of the cellular response is not protective but contributes by walling off the sites of infection to prevent dissemination and reactivation disease.
Reduced Virulence of Group A Streptococcal Tn
916
Mutants That Do Not Produce Streptolysin S
Authors: Betschel Stephen D. , Borgia Sergio M. , Barg Neil L. , Low Donald E. , De Azavedo Joyce C. S.
Published: December 2019

Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn
916
insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn
916
donor strain
Enterococcus faecalis
CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn
916
insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated
sagA
, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, gained weight during the first 24 h (+1.15 versus −1.16 g;
P
< 0.05) and had fewer necrotic lesions (0 versus 7;
P
= 0.0007). Animals infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus −0.66 g;
P
< 0.05) and produced fewer necrotic lesions (1 versus 8;
P
= 0.001). Revertants of the mutants in which Tn
916
had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn
916
mutations, are markedly less virulent than their isogenic parents.
Characteristics of Invasive Candidiasis in Gamma Interferon- and Interleukin-4-Deficient Mice: Role of Macrophages in Host Defense againstCandida albicans
Authors: Káposzta Rita , Tree Peter , Maródi László , Gordon Siamon
Published: December 2019

Murine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense againstCandida albicansand to characterize the tissue inflammatory reactions, with special reference to macrophages (Mφ). Knockout (KO) IFN-γ-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 108C. albicansblastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mφ. In the absence of IFN-γ,C. albicansinduced striking changes in the phenotype of alveolar Mφ and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mφ was shown only in GKO mice, and they producedCandida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-γ, but not IL-4, in host defense againstC. albicans.
Molecular Characterization of the Fragilysin Pathogenicity Islet of Enterotoxigenic
Bacteroides fragilis
Authors: Moncrief J. Scott , Duncan A. Jane , Wright Rhonda L. , Barroso Lisa A. , Wilkins Tracy D.
Published: December 2019

Enterotoxigenic strains of
Bacteroides fragilis
produce an extracellular metalloprotease toxin (termed fragilysin) which is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops. We report here that the fragilysin gene is contained within a small genetic element termed the fragilysin pathogenicity islet. The pathogenicity islet of
B. fragilis
VPI 13784 was defined as 6,033 bp in length and contained nearly perfect 12-bp direct repeats near its ends. Sequencing across the ends of the pathogenicity islet from two additional enterotoxigenic strains, along with PCR analysis of 20 additional enterotoxigenic strains, revealed that the islet is inserted at a specific site on the
B. fragilis
chromosome. The site of integration in three nontoxigenic strains contained a 17-bp GC-rich sequence which was not present in toxigenic strains and may represent a target sequence for chromosomal integration. In addition to the fragilysin gene, we identified an open reading frame encoding a predicted protein with a size and structural features similar to those of fragilysin. The deduced amino acid sequence was 28.5% identical and 56.3% similar to fragilysin and contained a nearly identical zinc-binding motif and methionine-turn region.
Agents That Inhibit Rho, Rac, and Cdc42 Do Not Block Formation of Actin Pedestals in HeLa Cells Infected with Enteropathogenic
Escherichia coli
Authors: Ben-Ami Goni , Ozeri Vered , Hanski Emanuel , Hofmann Fred , Aktories Klaus , Hahn Klaus M. , Bokoch Gary M. , Rosenshine Ilan
Published: December 2019

Enteropathogenic
Escherichia coli
(EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including
Clostridium difficile
toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.
Human Intestinal Epithelial Cells Respond toCryptosporidium parvumInfection with Increased Prostaglandin H Synthase 2 Expression and Prostaglandin E2and F2αProduction
Authors: Laurent Fabrice , Kagnoff Martin F. , Savidge Tor C. , Naciri Muriel , Eckmann Lars
Published: December 2019

Cryptosporidium parvumis an important cause of diarrhea in humans and several animal species. Prostaglandins play a central role in regulating intestinal fluid secretion in animal models of cryptosporidiosis, but their cellular sources and mechanisms of induction are unclear. Here, we show thatC. parvuminfection directly activates prostaglandin H synthase 2 expression and prostaglandin E2and F2αproduction in human intestinal epithelial cells.
Granulocytic Ehrlichiosis in Tick-Immune Guinea Pigs
Authors: Das Subrata , Deponte Kathleen , Marcantonio Nancy L. , Ijdo Jacob W. , Hodzic Emir , Katavolos Paula , Barthold Stephen W. , Telford Sam R. , Kantor Fred S. , Fikrig Erol
Published: December 2019

We investigated whether
Ixodes scapularis
-mediated host immunity interrupts transmission of the agent of human granulocytic ehrlichiosis (aoHGE) to guinea pigs. Ticks infected with aoHGE readily transmitted aoHGE to tick-immune guinea pigs, despite incomplete tick engorgement and host attachment. Although tick immunity can prevent Lyme borreliosis, protection is not afforded against granulocytic ehrlichiosis.
Molecular Characterization of the
Aeromonas hydrophila aroA
Gene and Potential Use of an Auxotrophic
aroA
Mutant as a Live Attenuated Vaccine
Authors: Hernanz Moral Carmen , del Castillo Emilio Flaño , Fierro Pilar López , Cortés Alberto Villena , Castillo Juan Anguita , Soriano Alberto Cascón , Salazar María Sánchez , Peralta Blanca Razquín , Carrasco Germán Naharro
Published: December 2019

The
aroA
gene of
Aeromonas hydrophila
SO2/2, encoding 5-enolpyruvylshikimate 3-phosphate synthase, was cloned by complementation of the
aroA
mutation in
Escherichia coli
K-12 strain AB2829, and the nucleotide sequence was determined. The nucleotide sequence of the
A. hydrophila aroA
gene encoded a protein of 440 amino acids which showed a high degree of homology to other bacterial AroA proteins. To obtain an effective attenuated live vaccine against
A. hydrophila
infections in fish, the
aroA
gene was inactivated by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of
A. hydrophila
AG2 by means of the suicide vector pSUP202. The
A. hydrophila
mutant AG2
aroA
::Ka
r
was highly attenuated when inoculated intraperitoneally into a rainbow trout, with a 50% lethal dose of >2 × 10
8
CFU. The mutants were not recoverable from the internal organs after 48 h postinoculation. Immunohistochemical studies demonstrated that immunopositive materials, but not whole cells, reacting with a polyclonal antiserum against
A. hydrophila
were present in the kidney and spleen 9 days postinjection. Vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection against the wild-type strain of
A. hydrophila.
cagA
-Positive
Helicobacter pylori
Populations in China and The Netherlands Are Distinct
Authors: van der Ende Arie , Pan Zhi-Jun , Bart Aldert , van der Hulst René W. M. , Feller Monique , Xiao Shu-Dong , Tytgat Guido N. J. , Dankert Jacob
Published: December 2019

The aim of this research was to study whether and to what extent Chinese
cagA
-positive
Helicobacter pylori
isolates differ from those in The Netherlands. Analysis of random amplified polymorphic DNA (RAPD)-PCR-assessed DNA fingerprints of chromosomal DNA of 24
cagA
-positive
H. pylori
isolates from Dutch (
n
= 12) and Chinese (
n
= 10) patients yielded the absence of clustering. Based on comparison of the sequence of a 243-nucleotide part of
cagA
, the Dutch (group I) and Chinese (group II)
H. pylori
isolates formed two separate branches with high confidence limits in the phylogenetic tree. These two clusters were not observed when the sequence of a 240-bp part of
glmM
was used in the comparison. The number of nonsynonymous substitutions was much higher in
cagA
than in
glmM
, indicating positive selection. The average levels of divergence of
cagA
at the nucleotide and protein levels between group I and II isolates were found to be high, 13.3 and 17.9%, respectively. Possibly, the pathogenicity island (PAI) that has been integrated into the chromosome of the ancestor of
H. pylori
now circulating in China contained a different
cagA
than the PAI that has been integrated into the chromosome of the ancestor of
H. pylori
now circulating in The Netherlands. We conclude that in China and The Netherlands, two distinct
cagA
-positive
H. pylori
populations are circulating.
Cytopathic Effects of the Major Surface Protein and the Chymotrypsinlike Protease ofTreponema denticola
Authors: Fenno J. Christopher , Hannam Pauline M. , Leung W. Keung , Tamura Muneaki , Uitto Veli-Jukka , McBride Barry C.
Published: December 2019

Prominent antigens ofTreponema denticolahave been suggested to be mediators of the cytopathic effects typically seen in periodontal disease. In the present study of theT. denticolamajor surface protein (Msp) and the surface-expressed chymotrypsinlike protease complex (CTLP), we characterized the ability of these proteins to adhere to and lyse epithelial cells. Msp and CTLP were closely associated in spirochete outer membranes. Purified Msp, both native and recombinant, and CTLP bound to glutaraldehyde-fixed periodontal ligament epithelial cells. Adherence of Msp was partially blocked by specific antibodies. Adherence of CTLP was partially blocked by serine protease inhibitors and was further inhibited by specific antibodies. Both native Msp and CTLP were cytotoxic toward periodontal ligament epithelial cells, and their cytotoxicity was inhibited by the same treatments that inhibited adherence. Msp, but not CTLP, lysed erythrocytes. Msp complex (partially purified outer membranes free of protease activity) was cytotoxic toward a variety of different cell types. Pore-forming activities of recombinant Msp in black lipid model membrane assays and in HeLa cell membranes were similar to those reported for the native protein, supporting the hypothesis that Msp cytotoxicity was due to its pore-forming activity.
Role of YopP in Suppression of Tumor Necrosis Factor Alpha Release by Macrophages duringYersiniaInfection
Authors: Boland Anne , Cornelis Guy R.
Published: December 2019

TheYersiniaplasmid-encoded Yop virulon enables extracellular adhering bacteria to deliver toxic effector proteins inside their target cells. It includes a type III secretion system (Ysc), at least two translocator proteins (YopB, YopD), and a set of intracellular Yop effectors (YopE, YopH, YopO, YopM, and YopP). Infection of macrophages with a wild-type strain leads to low levels of tumor necrosis factor alpha (TNF-α) release compared to infection with plasmid-cured strains, suggesting that the virulence plasmid encodes a factor impairing the normal TNF-α response of infected macrophages. This effect is correlated with the inhibition of the macrophage mitogen-activated protein kinase (MAPK) activities. To identify the Yop protein responsible for the suppression of TNF-α release, we infected J774A.1 and PU5-1.8 macrophages with a battery of knockoutYersinia enterocoliticamutants and we quantified the TNF-α released. Mutants affected in secretion (yscN), in translocation (yopBandyopD), or in synthesis of all the known Yop effectors (yopH,yopO,yopP,yopE, andyopMpolymutants) were unable to block the TNF-α response of the macrophages. In contrast, singleyopE,yopH,yopO, andyopMmutants behaved like the wild-type strain. AyopPmutant elicited elevated TNF-α release, and complementation of theyopPmutant or theyopeffector polymutant strain withyopPalone led to a drop in TNF-α release. In addition, YopP was also responsible for the inhibition of the extracellular signal-regulated kinase2 (ERK2) and p38 MAPK activities. These results show that YopP is the Yop effector responsible for theYersinia-induced suppression of TNF-α release by infected macrophages.
Validation of a Volunteer Model of Cholera with Frozen Bacteria as the Challenge
Authors: Sack David A. , Tacket Carol O. , Cohen Mitchell B. , Sack R. Bradley , Losonsky Genevieve A. , Shimko Janet , Nataro James P. , Edelman Robert , Levine Myron M. , Giannella Ralph A. , Schiff Gilbert , Lang Dennis
Published: December 2019

To evaluate a standardized inoculum of
Vibrio cholerae
for volunteer challenge studies, 40 healthy adult volunteers were challenged at three different institutions with a standard inoculum prepared directly from vials of frozen, virulent, El Tor Inaba
V. cholerae
N16961, with no further incubation. Groups of 5 volunteers, with each group including 2 volunteers with blood group O, were given a dose of 10
5
CFU, and 34 of the 40 volunteers developed diarrhea (mean incubation time, 28 h). Transient fevers occurred in 15 (37.5%) of the volunteers.
V. cholerae
was excreted by 36 of 40 volunteers. Five additional volunteers received 10
4
CFU, and four developed diarrhea but with a lower average purging rate than required for the model. Of the 40 volunteers, 37 developed rises in their vibriocidal and antitoxin titers similar to those in previous groups challenged with freshly harvested bacteria. We conclude that challenge with frozen bacteria results in a reproducible illness similar to that induced by freshly harvested bacteria. Use of this model should minimize differences in attack rates or severity when groups are challenged at different times and in different institutions.
Adherence of Human Vaginal Lactobacilli to Vaginal Epithelial Cells and Interaction with Uropathogens
Authors: Boris Soledad , Suárez Juan E. , Vázquez Fernando , Barbés Covadonga
Published: December 2019

Three strains of
Lactobacillus
, identified as
Lactobacillus acidophilus
,
Lactobacillus gasseri
, and
Lactobacillus jensenii
, were selected from among 70 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization or antagonism. All three self-aggregated and adhered to epithelial vaginal cells, displacing well-known vaginal pathogens, such as
G. vaginalis
, and inhibiting the growth in vitro of
Escherichia coli
and
Streptococcus agalactiae
. The surface components involved in self-aggregation appeared to be proteins for
L. gasseri
and lipoproteins for
L. acidophilus
and
L. jensenii
, as judged by susceptibility to treatment with appropriate degrading enzymes. The factors responsible for adherence to epithelial vaginal cells seemed to be glycoproteins (
L. acidophilus
and
L. gasseri
) and carbohydrate (
L. jensenii
). The receptors of the vaginal cells were glycolipids, which presumably were the targets of the competition observed between the lactobacilli and the pathogenic microbes.
Defined Deletion Mutants Demonstrate that the Major Secreted Toxins Are Not Essential for the Virulence of
Aeromonas salmonicida
Authors: Vipond Richard , Bricknell Ian R. , Durant Emma , Bowden Timothy J. , Ellis Anthony E. , Smith Mary , MacIntyre Sheila
Published: December 2019

The importance of the two major extracellular enzymes of
Aeromonas salmonicida
, glycerophospholipid:cholesterol acyltransferase (GCAT) and a serine protease (AspA), to the pathology and mortality of salmonid fish with furunculosis had been indicated in toxicity studies. In this study, the genes encoding GCAT (
satA
) and AspA (
aspA
) have been cloned and mutagenized by marker replacement of internal deletions, and the constructs have been used for the creation of isogenic
satA
and
aspA
mutants of
A. salmonicida
. A pSUP202 derivative (pSUP202sac) carrying the
sacRB
genes was constructed to facilitate the selection of mutants. The requirement of serine protease for processing of pro-GCAT was demonstrated. Processing involved the removal of a short internal fragment. Surprisingly, pathogenicity trials revealed no major decrease in virulence of the
A. salmonicida ΔsatA
::
kan
or
A. salmonicida ΔaspA
::
kan
mutants compared to the wild-type parent strains when Atlantic salmon (
Salmo salar
L.) were challenged by intraperitoneal injection. Moreover, using a cohabitation model, which more closely mimics the natural disease, there was also no significant decrease in the relative cumulative mortality following infection with either of the deletion mutants compared to the parent strain. Thus, although these two toxins may confer some competitive advantage to
A. salmonicida
, neither toxin is essential for the very high virulence of
A. salmonicida
in Atlantic salmon. This first report of defined deletion mutations within any proposed extracellular virulence factor of
A. salmonicida
raises crucial questions about the pathogenesis of this important fish pathogen.
Binding of
Porphyromonas gingivalis
Fimbriae to Proline-Rich Glycoproteins in Parotid Saliva via a Domain Shared by Major Salivary Components
Authors: Amano Atsuo , Shizukuishi Satoshi , Horie Hiroshi , Kimura Shigenobu , Morisaki Ichijiro , Hamada Shigeyuki
Published: December 2019

Porphyromonas gingivalis
, a putative periodontopathogen, can bind to human saliva through its fimbriae. We previously found that salivary components from the submandibular and sublingual glands bind to
P. gingivalis
fimbriae and that acidic proline-rich protein (PRP) and statherin function as receptor molecules for fimbriae. In this study, we investigated the fimbria-binding components in parotid saliva. Fractionated human parotid saliva by gel-filtration chromatography was immobilized onto nitrocellulose membranes for the overlay assay following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The salivary components on the membrane were allowed to interact with fimbriae purified from
P. gingivalis
ATCC 33277, and the interacted fimbriae were probed with anti-fimbria antibodies. The fimbriae were shown to bind to two forms of proline-rich glycoproteins (PRGs) as well as to acidic PRPs and statherin. Moreover, fimbriae bound to several components of smaller molecular size which appeared to be acidic PRP variants and basic PRPs. Fimbriae bound strongly to the purified PRGs adsorbed onto hydroxyapatite (HAP) beads. In contrast, PRGs in solution failed to inhibit the fimbrial binding to the immobilized PRGs on the HAP beads. These findings suggest that the appearance of binding site(s) of PRGs can be ascribed to their conformational changes. We previously identified the distinct segments within PRP and statherin molecules that are involved in fimbrial binding. The peptides analogous to the binding regions of PRP and statherin (i.e., PRP-C and STN-C) markedly inhibit the binding of fimbriae to PRP and statherin immobilized on the HAP beads, respectively. The PRP-C significantly inhibited the binding of fimbriae to PRG-coated HAP beads as well as to PRP on HAP beads. The peptide did not affect the binding of fimbriae to statherin, whereas the STN-C showed no effect on the fimbrial binding to PRPs or PRGs. In the overlay assay, the PRP-C clearly diminished the interactions between the fimbriae and the various salivary components, including PRPs, the PRGs, and the components with smaller molecular sizes but not statherin. These results strongly suggest that fimbriae bind to salivary components (except statherin) via common peptide segments. It is also suggested that fimbriae bind to saliva through the two distinct binding domains of receptory salivary components: (i) PRGs and PRPs and (ii) statherin.
A 70-Kilodalton Recombinant Heat Shock Protein ofCandida albicansIs Highly Immunogenic and Enhances Systemic Murine Candidiasis
Authors: Bromuro Carla , La Valle Roberto , Sandini Silvia , Urbani Francesca , Ausiello Clara M. , Morelli Luisella , Féd’ostiani Cristiana , Romani Luigina , Cassone Antonio
Published: December 2019

The 70-kDa recombinantCandida albicansheat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-γ), IL-12, and IL-10 but not IL-4 production by CD4+lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-γ was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-γ upon in vitro stimulation of CD4+cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement ofCandidainfection seemed to occur in the mouse model used.
Use of a
Mycobacterium tuberculosis
H37Rv Bacterial Artificial Chromosome Library for Genome Mapping, Sequencing, and Comparative Genomics
Authors: Brosch Roland , Gordon Stephen V. , Billault Alain , Garnier Thierry , Eiglmeier Karin , Soravito Catherine , Barrell Bart G. , Cole Stewart T.
Published: December 2019

The bacterial artificial chromosome (BAC) cloning system is capable of stably propagating large, complex DNA inserts in
Escherichia coli
. As part of the
Mycobacterium tuberculosis
H37Rv genome sequencing project, a BAC library was constructed in the pBeloBAC11 vector and used for genome mapping, confirmation of sequence assembly, and sequencing. The library contains about 5,000 BAC clones, with inserts ranging in size from 25 to 104 kb, representing theoretically a 70-fold coverage of the
M. tuberculosis
genome (4.4 Mb). A total of 840 sequences from the T7 and SP6 termini of 420 BACs were determined and compared to those of a partial genomic database. These sequences showed excellent correlation between the estimated sizes and positions of the BAC clones and the sizes and positions of previously sequenced cosmids and the resulting contigs. Many BAC clones represent linking clones between sequenced cosmids, allowing full coverage of the H37Rv chromosome, and they are now being shotgun sequenced in the framework of the H37Rv sequencing project. Also, no chimeric, deleted, or rearranged BAC clones were detected, which was of major importance for the correct mapping and assembly of the H37Rv sequence. The minimal overlapping set contains 68 unique BAC clones and spans the whole H37Rv chromosome with the exception of a single gap of ∼150 kb. As a postgenomic application, the canonical BAC set was used in a comparative study to reveal chromosomal polymorphisms between
M. tuberculosis
,
M. bovis
, and
M. bovis
BCG Pasteur, and a novel 12.7-kb segment present in
M. tuberculosis
but absent from
M. bovis
and
M. bovis
BCG was characterized. This region contains a set of genes whose products show low similarity to proteins involved in polysaccharide biosynthesis. The H37Rv BAC library therefore provides us with a powerful tool both for the generation and confirmation of sequence data as well as for comparative genomics and other postgenomic applications. It represents a major resource for present and future
M. tuberculosis
research projects.
Isolation of the Third Capsule-Associated Gene,
CAP60
, Required for Virulence in
Cryptococcus neoformans
Authors: Chang Y. C. , Kwon-Chung K. J.
Published: December 2019

A polysaccharide capsule is one of the most important virulence factors for the pathogenic fungus
Cryptococcus neoformans
. We previously characterized two capsule-associated genes,
CAP59
and
CAP64
. To further dissect the molecular mechanism of capsule synthesis, 16 acapsular mutants induced by 4-nitroquinoline-1-oxide were obtained. The acapsular phenotype of one of these mutants was complemented. The cloned gene was designated
CAP60
, and deletion of this newly described capsule-associated gene resulted in an acapsular phenotype. The proposed 67-kDa Cap60p contains 592 amino acids and appears to have a putative transmembrane domain close to the N terminus. DNA sequence analysis revealed that
CAP60
has similarity to
CAP59
at the center portion of its coding regions. Contour-clamped homogeneous electric field blot analysis suggested that these two genes are on the same chromosome.
CAP60
and
CAP59
, however, could not be functionally substituted for each other by direct complementation or by domain swap experiments. In addition,
CAP60
is closely linked to a gene which is similar to a cellulose growth-specific gene of
Agaricus bisporus
,
CEL1
. Immunogold electron microscopy studies of the epitope-tagged
CAP60
gene revealed that Cap60p was primarily localized to the nuclear membrane. Animal model studies indicated that
CAP60
is essential for virulence. Thus,
CAP60
is required for both capsule formation and virulence.
Potential of a Novel Protein, OMP26, from Nontypeable
Haemophilus influenzae
To Enhance Pulmonary Clearance in a Rat Model
Authors: Kyd Jennelle M. , Cripps Allan W.
Published: December 2019

A major outer membrane protein band of approximately 25 to 27 kDa is commonly observed in strains of
Haemophilus influenzae
. This study has investigated the potential of a 26-kDa protein (OMP26) from nontypeable
H. influenzae
(NTHI) as a vaccine candidate. OMP26 was used to immunize rats via intestinal Peyer’s patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain. Significant levels of anti-OMP26 were found in the serum and bronchoalveolar lavage from immunized rats, and isotypes of immunoglobulin G (IgG) were also measured in serum. Analysis of IgG isotypes present in serum following OMP26-immunization suggest that predominantly a T-helper 1-type response was induced. The OMP26 protein was amino-terminally sequenced and found to have no homology with the P5 of
H. influenzae
type b P5 or the fimbrin protein of NTHI, both can migrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis at similar molecular masses but OMP26 has 100% homology with a segment of the
H. influenzae
Rd genome. The results of this study suggest that OMP26 may be a suitable vaccine candidate against NTHI infection and warrants continued investigation and characterization.
Differential Effects of Gamma Interferon onChlamydia trachomatisGrowth in Polarized and Nonpolarized Human Epithelial Cells in Culture
Authors: Kane Colleen D. , Byrne Gerald I.
Published: December 2019

The effects of gamma interferon (IFN-γ) onChlamydia trachomatisgrowth in polarized epithelial cells were examined. The range of IFN-γ concentrations causing aberrant chlamydial growth was wider in polarized than in nonpolarized cultures. Results indicate that chlamydial growth modulation in polarized cells readily leads to persistence and better reflects in vivo conditions.
VirulentBrucella abortusPrevents Lysosome Fusion and Is Distributed within Autophagosome-Like Compartments
Authors: Pizarro-Cerdá Javier , Moreno Edgardo , Sanguedolce Veronique , Mege Jean-Louis , Gorvel Jean-Pierre
Published: December 2019

Virulent and attenuatedBrucella abortusstrains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenicBrucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting thatBrucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes.
Activity of Protegrins against Yeast-Phase
Candida albicans
Authors: Cho Yoon , Turner Jeffrey S. , Dinh Nhu-Nguyen , Lehrer Robert I.
Published: December 2019

We used a two-stage radial diffusion assay to perform a structure-activity study of the antifungal effects of protegrin-1 (PG-1) on yeast-phase
Candida albicans
. While doing so, we computed MICs from the radial diffusion assay data by three methods and compared the respective values with results from colony count and broth microdilution assays. This allowed us to identify several technical modifications that improved the sensitivity and accuracy of radial diffusion assays. We found that both PG-1 and enantiomeric PG-1 (composed exclusively of
d
-amino acids) were potently fungicidal for yeast-phase
C. albicans
. The protegrins PG-2, -3, and -5, but not PG-4, were as effective as PG-1. At least one intramolecular disulfide bond was required to retain optimal candidacidal activity at physiological NaCl concentrations. Truncated variants of PG-1 that lacked its first four residues showed decreased candidacidal activity, although their activity against bacteria was substantially intact. Altering the β-turn region (residues 9 to 12) of PG-1 or its variants further decreased candidacidal activity. These studies suggest that only 12 residues are needed to endow protegrin molecules with strong antibacterial activity and that at least 4 additional residues are needed to add potent antifungal properties. Thus, the 16-residue protegrin PG-2 likely represents the minimal structure needed for broad-spectrum antimicrobial activity encompassing bacteria and fungi.
The
Vibrio cholerae
Mannose-Sensitive Hemagglutinin Is the Receptor for a Filamentous Bacteriophage from
V. cholerae
O139
Authors: Jouravleva Elena A. , McDonald Gregory A. , Marsh Jane W. , Taylor Ronald K. , Boesman-Finkelstein Mary , Finkelstein Richard A.
Published: December 2019

We previously isolated from a 1994 isolate of
Vibrio cholerae
O139 a filamentous lysogenic bacteriophage, choleraphage 493, which inhibits pre-O139 but not post-O139 El Tor biotype
V. cholerae
strains in plaque assays. We investigated the role of the mannose-sensitive hemagglutinin (MSHA) type IV pilus as a receptor in phage 493 infection. Spontaneous, Tn
5
insertion, and
mshA
deletion mutants are resistant to 493 infection. Susceptibility is restored by
mshA
complementation of deletion mutants. Additionally, the 493 phage titer is reduced by adsorption with MSHA-positive strains but not with a Δ
mshA1
strain. Monoclonal antibody against MSHA inhibits plaque formation. We conclude that MSHA is the receptor for phage 493. The emergence and decline of O139 in India and Bangladesh are correlated with the susceptibility and resistance of El Tor strains to 493. However,
mshA
gene sequences of post-O139 strains are identical to those of susceptible pre-O139 isolates, indicating that phage resistance of El Tor is not due to a change in
mshA
. Classical biotype strains are (with rare exceptions) hemagglutinin negative and resistant to 493 in plaque assays. Nevertheless, they express the
mshA
pilin gene. They can be infected with 493 and produce low levels of phage DNA, like post-O139 El Tor strains. Resistance to 493 in plaque assays is thus not equivalent to resistance to infection. The ability of filamentous phages, such as 493, to transfer large amounts of DNA provides them, additionally, with the potential for quantum leaps in both identity and pathogenicity, such as the conversion of El Tor to O139.